Anti-androgens entering the aquatic environment, e.g., by effluents from wastewater treatment plants or agricultural settings are contributing to endocrine disruption in wildlife and humans. Due to the simultaneous presence of agonistic compounds, common in vitro bioassays can underestimate the risk posed by androgen antagonists.
Trang 1Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/chroma
Carolin Riegraf1, 2, Anna Maria Bell2, Marina Ohlig, Georg Reifferscheid,
Sebastian Buchinger∗
Federal Institute of Hydrology, Am Mainzer Tor 1, 56068 Koblenz, Germany
a r t i c l e i n f o
Article history:
Received 28 April 2022
Revised 13 October 2022
Accepted 17 October 2022
Available online 19 October 2022
Keywords:
Anti-androgenicity
Androgenicity
Cytotoxicity
High-performance thin-layer
chromatography
Effect-directed analysis
a b s t r a c t
Anti-androgensenteringtheaquaticenvironment,e.g.,byeffluentsfromwastewatertreatmentplantsor agriculturalsettingsarecontributingtoendocrinedisruptioninwildlifeandhumans.Duetothe simul-taneouspresenceofagonisticcompounds,common in vitro bioassayscanunderestimatetheriskposed
byandrogenantagonists.Ontheotherhand,cytotoxiceffectsmightleadtofalsepositiveassessmentsof anti-androgeniceffectsinconventionalbioassays.Inthepresentstudy, acombinationofnormalphase high-performancethin-layerchromatography(NP-HPTLC)withayeast-basedreportergeneassayis es-tablishedforthe detectionofanti-androgenicityas apromisingtool toreduceinterferencesof andro-genicandanti-androgeniccompoundspresentinthesamesample.Toavoidamisinterpretationof anti-androgenicity with cytotoxiceffects, cell viability was assessedinparallel onthe same plateusing a resazurinviability assayadaptedtoHPTLCplates.Themethodwascharacterizedbyestablishing dose-responsecurvesforthemodelcompoundsflutamideandbisphenolA.Calculatedeffectivedosesat10% (ED10)were27.9± 1.3ngzone−1forflutamideand20.1± 5.1ngzone−1forbisphenolA.Successful dis-tinctionbetweenanti-androgenicityandcytotoxicitywasexemplarilydemonstratedwith4-nitroquinoline 1-oxide.Asaproofofconcept, thedetectionandquantificationofanti-androgenicityinanextractofa landfillleachateisdemonstrated.ThisstudyshowsthatthehyphenationofHPTLCwiththeyeast anti-androgenscreenisamatrix-robust,cost-efficientand fastscreeningtoolforthesensitiveand simulta-neousdetectionofanti-androgenicandcytotoxiceffectsinenvironmentalsamples.Themethodoffersa widerangeofpossibleapplicationsinenvironmentalmonitoringandcontributestotheidentificationof anti-androgenicitydriversinthecourseofaneffect-directedanalysis
© 2022 The Author(s) Published by Elsevier B.V ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/)
1 Introduction
Many different environmental contaminants are present in the
aquatic environment continuously released by wastewater treat-
ment plants (WWTPs), agricultural settings or aquacultures [ 1, 2]
Among these are compounds, which can have adverse effects on
the endocrine system, so called endocrine disrupting compounds
(EDCs) In particular, many previous studies were focused on EDCs
that trigger estrogenicity via estrogen receptor-mediated signaling
pathways associated with adverse effects such as feminization and
vitellogenin production in male fish [ 3, 4]
∗ Corresponding author at: Federal Institute of Hydrology, Department G3 - Bio-
chemistry, Ecotoxicology, Am Mainzer Tor 1, 56068 Koblenz, Germany
E-mail address: Buchinger@bafg.de (S Buchinger)
1 Present address: Swiss Centre for Applied Ecotoxicology, Überlandstrasse 133,
8600 Dübendorf, Switzerland
2 These authors contributed equally to this work
Besides estrogenicity elicited by (xeno-)estrogens, several stud- ies worldwide revealed the presence of compounds influencing the androgen receptor, e.g., in effluents of paper and pulp industries [5], rangeland grazing and beef cattle feedlot [ 6, 7], leather fab- rication [8], and WWTPs [ 9, 10] or in river sediments [11] These compounds can act as receptor agonists or antagonists, which ei- ther activate (androgenic compounds) or inhibit (anti-androgenic compounds) hormonal androgen receptors, respectively, and hence, the endogenous hormonal activity As consequence, masculiniza- tion and effects on the immune system of aquatic biota caused by androgens were observed [12] In contrast, anti-androgenic com- pounds induced inter alia feminization of non-mammalian verte- brate males, changes in gender ratio as well as inhibited oogenesis and spermatogenesis [13]
Whereas estrogenic and androgenic activities were found to
be largely eliminated by biological and advanced treatment of wastewater, antagonistic activities were only removed up to 50%
https://doi.org/10.1016/j.chroma.2022.463582
0021-9673/© 2022 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )
Trang 2by biological treatment and also not further by advanced treat-
ment [14] Thus, there is a need to further investigate antagonistic
effects such as anti-androgenicity, which could contribute besides
steroidal estrogens to endocrine disruption in wildlife [ 9, 15]
Several compounds are known for their anti-androgenic activity,
e.g., natural and synthetic steroids such as androstenone [16], nan-
drolone [16]or cyproterone acetate [13], but also industrial com-
pounds such as bisphenol A and butyl benzyl phthalate [17], pet-
rogenic naphthenic acids [18], the germicides chlorophene and tri-
closan [10], the fungicide vinclozolin [13], polycyclic musks [16]or
Coumarin 47 [19], and other consumer products [10] Nevertheless,
the identification of anti-androgenic effect-inducing compounds
remains in many cases challenging [19]
Effect-based methods (EBMs) are tools to assess the overall ef-
fect potential of a sample taking into account the contribution of
unknown compounds Several EBMs are available for the detection
of (anti-)androgenic effects Recombinant cells such as yeast [ 17,
20] and mammalian cell lines [ 21, 22] are frequently used in re-
spective receptor-based reporter gene assays These in vitro assays
usually performed in microtiter well plates assess the overall ef-
fect potential of a sample including potential mixture effects De-
pending on the underlying objective this can be a desired feature,
however, the presence of both agonistic and antagonistic could
mask their mutual activity potentially leading to false conclusions
[23] Furthermore, cytotoxic effects might interfere with the detec-
tion of a specific effect in microwell-based assays [24] Hashmi,
et al [25] concluded in a recent study from 2020 that there is
a need “to better understand the occurrence of EDCs and mask-
ing compounds in different lipophilicity windows, to finally reduce
fractionation requirements for monitoring to a smart clean-up.” In
this respect the direct combination of high-performance thin-layer
chromatography (HPTLC) with in vitro-bioassays provides an effi-
cient platform as a matrix-robust, cost-efficient and fast screening
approach that can guide subsequent in-depth EDA [26] For this ap-
proach samples are separated on a HPTLC plate into different frac-
tions prior to performing the EBM directly on the surface of the
HPTLC plate After exposure of the cells, potential effect-inducing
fractions are observable and can be quantified This approach was
successfully applied for the detection of compounds with specific
modes of action such as estrogenicity in personal care products
[27] or food contact materials [28], androgenicity in WWTP ef-
fluents [29] or photosystem II inhibition in surface water extracts
[30] Recently, Klingelhöfer, et al [31]published a reversed phase
HPTLC planar yeast anti-androgen screen (RP-HPTLC-p-YAAS) for
the detection of anti-androgenic compounds using lacZ as reporter
gene
The objective of the present study was to develop a p-YAAS
on normal phase HPTLC plates as a complementary approach to
the reversed phase HPTLC to broaden the applicability, further in-
crease the sensitivity of the bioassay and simultaneously enable a
quantification of antagonistic effects In addition, a combination of
the p-YAAS with a test for cell viability was implemented to dis-
tinguish between anti-androgenic and cytotoxic compounds on the
same HPTLC plate The developed methodology was examined and
optimized in terms of effective doses (ED) and repeatability using
mixtures of reference compounds As proof of principle for an ap-
plication to complex matrices, the methodology was used to assess
the anti-androgenic potential of a landfill leachate extract
2 Materials and methods
2.1 Chemicals
The model compounds testosterone (CAS: 58-22-0), flutamide
(CAS: 13311-84-7), bisphenol A (BPA, CAS: 80-05-7, ≥99%) and
4-nitroquinoline 1-oxide (4-NQO, CAS: 56-57-5, ≥98%) were ob-
tained from Merck in the highest purity commercially available The solvents ethanol ( ≥99.8%), chloroform (99.0 99.4%), n-hexane (99.0%), methanol (99.9%) and petroleum fraction (bp 65 – 100 °C) were purchased from Merck Ethyl acetate (EtAc, 99.8%) was ac- quired from LGC Standards Resazurin sodium salt (CAS: 62758-13- 8) was obtained from Merck The components for growth- and ex- posure medium were obtained from Merck in the highest grade commercially available
2.2 Media and solutions
Stock solutions of model compounds were prepared in ethanol The stock solution of resazurin was prepared in double distilled water and stored at 4 °C in the dark
The growth medium contained 6.7 g L −1 yeast nitrogen base without amino acids, 20 g L −1glucose, and the appropriate amino acids, i.e adenine (20 mg L −1), arginine (20 mg L −1), aspartic acid (100 mg L −1), glutamic acid (100 mg L −1), histidine (20 mg L −1), isoleucine (30 mg L −1), leucine (100 mg L −1), lysine (30 mg L −1), methionine (20 mg L −1), phenylalanine (50 mg L −1), serine (400
mg L −1), threonine (200 mg L −1), tyrosine (30 mg L −1) and valine (150 mg L −1), in double-distilled water The exposure medium was five-times higher concentrated than the growth medium and addi- tionally supplemented with 8 μL mL −1 CuSO 4-solution (2.5 g L −1
in double distilled water)
The lacZ reaction mixture consisted of 10 mL lacZ-buffer (10.67
g L −1Na 2HPO 4 · 2 H 2O, 0.75 g L −1KCl, 0.25 g L −1MgSO 4 · 7 H 20, 5.5 g L −1 NaH 2PO 4 · H2O, and 1 g L −1 sodium dodecyl sulfate) and 0.5 mg mL −1 of 4-methylumbelliferyl- β-D-galactopyranoside (MUG, CAS: 6160-78-7, dissolved in dimethyl sulfoxide (DMSO, CAS: 67-68-5))
2.3 Landfill leachate extract
Leachate of a mixed deposition site was collected as a grab sample at a landfill site in Germany prior to leachate water treat- ment Until 2005, this landfill was also used for deposition of do- mestic and bulky waste, from 2006 only commercial and construc- tion waste as well as sewage and industrial sludges were disposed
of there The sample was collected, filtered and 200-fold enriched
by solid phase extraction as described in detail in Riegraf, et al [32] Sample extract (1 mL) in methanol was stored at – 20 °C in 1.5 mL amber glass vials until use
2.4 Chromatographic separation
HPTLC was performed on 20 ×10 cm silica gel 60 F 254 HPTLC plates (Merck, Germany) HPTLC plates were pre-washed by chro- matographic development with methanol to 5 mm below the rim
in a TLC-developing-chamber (CAMAG) and subsequently activated
at 110 °C for 30 min in an oven prior to use [33] Dilutions of model compounds or sample extracts were applied as 5 mm band and 5 ×3 mm area, respectively, at 8 mm distance from the lower edge of the pre-washed HPTLC plates using an Automatic TLC Sam- pler 4 (ATS 4, CAMAG) A two-step chromatographic separation was performed using an Automated Multiple Development Sys- tem (AMD 2, CAMAG) with 1.) methanol up to 20 mm and 2a.) chloroform:EtAc:petroleum fraction (55:20:25, V:V:V, modified af- ter Cimpoiu, et al [34]) for separating flutamide and BPA or sample extracts or 2b.) EtAc:n-hexane (50:50, V:V) for the separation of flutamide and testosterone up to 90 mm [32] All automated CA- MAG devices were operated under the software visionCATS (ver- sion 2.5 SP1, CAMAG)
2
Trang 32.5 HPTLC-based planar yeast (anti-)androgen screen
Effects on the androgen receptor were evaluated using the an-
drogenic test strain BJ1991 derived from Saccharomyces cerevisiae
(MATa pep4-3, prbl-1122, ura3-52, leu2, frpl, GAL) [ 17, 35] The
preparation of cells for the planar androgen bioassay was de-
scribed elsewhere [29] Briefly, cells were cultivated overnight (20
mL growth medium inoculated with 1 mL cryogenic BJ1991 yeast
suspension) at 30 °C on a shaker (IKA® KS 30 0 0 i control, orbital
shaking at 200 rpm) Subsequently, cells were pelleted by centrifu-
gation (Hettich® Universal 320R, 2500 g for 10 min) and resus-
pended in fresh exposure medium The cell density was adjusted
to 1500 ± 50 FAU for automatic spray application, calibrated ac-
cording to ISO 7027-1 [36]at a wavelength of 600 nm using a plate
reader (Tecan Infinite® 200 PRO)
The planar yeast androgen screen (p-YAS) was performed as de-
scribed in Riegraf, et al [29] In brief, the adjusted cell suspension
was sprayed on the HPTLC plate using a HPTLC derivatizer (CA-
MAG) operated in a closed system (application volume: 3 mL, noz-
zle: yellow, spraying level: 5) For exposure, the HPTLC plates were
placed in a plastic box containing a paper towel soaked with 5 mL
double-distilled water and incubated at 30 °C and 90% relative hu-
midity for 20 h (NuAire CO 2-incubator with humidity control, NU-
5820E) After incubation, HPTLC plates were dried with cold air for
5 min using a fan Meanwhile, the lacZ reaction mixture was pre-
pared and sprayed on the dried HPTLC plate using a HPTLC deriva-
tizer (CAMAG, application volume: 2.5 mL, nozzle: green, spraying
level: 5) Subsequently, the HPTLC plate was placed in a plastic box
without lid, which in turn was placed in an incubator at 37 °C for
15 min to give time to the enzymatic reaction
For the detection of anti-androgenicity, a planar yeast anti-
androgen screen (p-YAAS) was developed by adapting the p-
YAS procedure described as follows A testosterone stock solution
in ethanol (2 mg mL −1) was diluted 1:200 with 10x exposure
medium Just before the exposure of the cells to the chromato-
graphically separated sample components on the HPTLC plate, the
adjusted yeast suspension was spiked with testosterone to a final
concentration of 50 ng mL −1 This yeast suspension was immedi-
ately sprayed on the HPTLC plate using a HPTLC derivatizer (CA-
MAG) operated in a closed system (application volume: 3mL, noz-
zle: yellow, spraying level: 5) Then, the procedure of the p-YAS as
described above was pursued
Agonistic and antagonistic potentials of the separated com-
pounds were detected qualitatively under UV light at a wavelength
of λ= 366 nm and exposure times of 550 ms, 20 0 0 ms as well
as automatic settings using the TLC Visualizer 2 (CAMAG) Further-
more, signals were documented using a TLC Scanner 4 (CAMAG) at
λex = 320 nm (deuterium lamp) and a cut-off filter of 400 nm
2.6 HPTLC-based planar resazurin cell viability assay for the
detection of yeast cytotoxicity
The planar assay for cell viability can be either performed on
the same HPTLC plate as the p-YAAS after the application of the
lacZ reaction mixture or on a separate HPTLC plate treated identi-
cally but without applying lacZ reaction mixture After drying the
plates with cold air for 5 min, 3 mL of resazurin solution (0.1 g
L −1 in double distilled water) was sprayed on the HPTLC plate us-
ing a HPTLC derivatizer (CAMAG, nozzle: green, spraying level: 5)
The HPTLC plate was incubated at 30 °C and 90% relative humid-
ity for 30 min After the incubation, signals visualized as dark rose
spots on a colorless background were captured at white light (di-
rect light, transmitted light and a combination of the both) using
the TLC Visualizer 2 (CAMAG) Subsequently, HPTLC plates were
dried again with cold air for 5 min, followed by a second doc-
umentation of the signals at white light Moreover, signals were
captured by scanning densitometry using the TLC Scanner 4 (CA- MAG) in absorption mode at λ= 575 nm under light emitted by a deuterium and a halogen-tungsten lamp without applying a filter
2.7 Data processing and statistical analysis
Excel® and R 3.5.2 [37], in particular the ‘drc‘ [38]and the ‘gg- plot2‘ [39] packages, were used for data processing and statistical analysis Peak areas of signals were extracted from the scan chro- matograms and expressed as arbitrary units (AU) Dose-response curves for model compounds were established by regression anal- ysis A four-parameter log-logistic function [40] was used to fit averaged obtained data from up to five replicates to a sigmoidal curve This dose-response curve served as basis for the calculation
of ED10 and ED50
The anti-androgenic effect of the landfill leachate was quan- tified by the calculation of biological equivalence concentrations (BEQs) by relating the observed effects caused by the antagonistic fractions to the dose-response data of the model compounds BPA and flutamide The resulting values reflect the amount of BPA and flutamide producing the same effect as the sample or their frac- tions of unknown composition considering the enrichment factor, pre-dilution and application volumes
3 Results
3.1 Development of p-YAAS on normal phase silica gel HPTLC plates
The YAAS conducted in 96-well plates served as starting point for the development of the p-YAAS Similarly to the YAAS, a high background signal is created induced by an agonist spiked to the applied yeast suspension Antagonistic compounds can then be de- tected based on fluorescence signal suppression In a first step, the engineered yeast cells were exposed on a HPTLC plate to var- ious amounts of the androgen receptor (AR) agonist testosterone
to adjust the background level for an optimal detection of antag- onistic effects visible as a suppression of the fluorescence signal Concentrations of 50, 100, 150, 200 and 250 ng mL −1testosterone spiked to the applied yeast suspension were tested Dose-response curves of the anti-androgenic model compounds flutamide and BPA were established by spot application without chromatographic de- velopment using the different testosterone concentrations The de- tectable anti-androgenic effects decreased with increasing spike concentration (see Fig S1) while the intensity of cytotoxic signals was not affected by the background agonist concentration (see Fig S2) A testosterone spike of 50 ng mL −1 led to the lowest ED50- values and was thus chosen as the final agonist spike concentra- tion
After the successful development of the p-YAAS procedure without chromatographic development ( Fig 1a.)), mixtures of model compounds were applied and chromatographically sepa- rated The mobile phase composition was adapted to reach a com- plete separation of the model compounds The final solvent com- position for separating flutamide and BPA consisted of a focus- ing step using 100% methanol followed by a separation step us- ing chloroform:EtAc:petroleum fraction (55:20:25, V:V:V, modified after Cimpoiu, et al [34])
A signal suppression in a dose responding manner with the separated model compounds flutamide and BPA were observed (Fig S3) The respective dose-response curves are shown in Fig.1a.) without and Fig.1b.) with chromatographic development The obtained dose response curves were used to calculate effective doses ( Table 1) Established ED10 values were lower with chro- matographic development compared to tests without chromato- graphic development The ED10 value for BPA with 35.1 ± 3.5
ng spot −1 was lower compared to the ED10 of flutamide with an
Trang 4Fig 1 Planar yeast anti-androgen screen (p-YAAS) a.) without and b.) with chromatographic separation of the model compounds bisphenol A and flutamide A two-step
chromatographic development was performed using methanol and chloroform:EtAc:petroleum fraction (55:20:25, V:V:V, modified after Cimpoiu, et al [31] ) In the graphs, mean signal intensities determined by the peak area are plotted against the applied amount of model compound Error bars show the respective standard deviation with a.)
n = 4 and b.) n = 3
Table 1
Effective doses 10% (ED10) and 50% (ED50) of flutamide and bisphenol A in the
planar yeast anti-androgen screen (p-YAAS) with and without chromatographic de-
velopment Shown are mean values ± standard deviation The number of replicates
was n without = 4 and n with = 3
Compound Effective dose Chromatographic development
without (ng spot −1 ) with (ng spot −1 )
ED10 of 46.3 ± 4.7 ng spot −1 ( Table 1) However, the ED50 val-
ues showed a different picture with lower values determined for
flutamide in comparison to BPA without chromatographic develop-
ment
3.2 Separation of anti-androgenic and androgenic compounds for
interference reduction
One main advantage of performing the YAAS on HPTLC plates
is the possibility to separate anti-androgenic and androgenic com-
pounds to reduce interferences of agonistic and antagonistic effects
that might result in a mutual masking of these specific effects A
proof of principle for the analysis of androgenic affects in the pres-
ence of an anti-androgen and vice versa was done by the analysis
of a mixture of testosterone and flutamide in a concentration ratio
of 1 to 1500 This mixture was first analyzed by the classic 96-well
based YAS and YAAS A significant reduction of agonistic and vice
versa antagonistic effects in the mixture was observed in the 96-
well based assays compared to the application of testosterone (Fig
S4a.)) and flutamide (Fig S4b.)) alone
Aliquots of the same mixture were applied in different volumes
on split HPTLC plates that were directed to the p-YAS and the p-
YAAS, respectively In Fig.2, the results of the p-YAS are shown on
the left side and the results of the p-YAAS are shown on the right
side By the comparison of signals detected in the mixture and
respective positive controls, i.e testosterone and flutamide alone,
it becomes evident that a distinction of agonistic and antagonis-
tic effects is possible by the HPTLC-based versions of the YAS and
the YAAS In case of the p-YAAS slightly increased signal intensities
are visible at the testosterone-specific migration distance when the
mixture is analyzed
3.3 Distinction between antagonistic and cytotoxic effects by means
of a resazurin cell viability assay
For a correct interpretation of antagonistic effects in reporter gene assays, it is of high importance to distinguish specific in- hibitory effects from a general cytotoxicity that also results in sig- nal suppression In 96-well plate assays, this is often done by as- sessing cellular growth, which is not possible on a HPTLC plate Therefore, a HPTLC-based assay for cell viability was integrated into the overall procedure This assay is based on the character- istic of viable yeast cells to irreversibly reduce the redox dye re- sazurin (orange at pH < 6.5 and dark violet at pH > 6.5) into the pink, fluorescent product (resorufin) and further to the color- less dihydroresorufin NAD(P)H generated by catabolic reactions in the cytoplasm and the mitochondria of living cells serves as the reducing agent for this reaction Thus, the viability of the yeast cells on the HPTLC plate can be assessed visually after the appli- cation of resazurin by a discoloration of the HPTLC plate at po- sitions with metabolically active cells In contrast, depending on the pH value and moisture content of the HPTLC plate, cytotoxic effects become visible as orange, rose, violet or blue spots This general principle had been previously adapted to the application
on HPTLC plates to detect the antimicrobial activity of peptides [41] and plant extracts [42] Furthermore, resazurin was used to confirm the results of a newly developed planar cytotox CALUX bioassay [43]
In line with the current study, several parameters for the on- plate resazurin assay were optimized First, the application of re- sazurin was improved in terms of application volume, nozzle size for spraying and concentration For this purpose, six different re- sazurin concentrations spanning a range of 0.1 – 5 g L −1 were applied on a HPTLC plate on which different amounts of the cy- totoxic 4-NQO were applied With increasing resazurin concentra- tion, the non-toxic area turned orange to brownish while the cy- totoxic signals stand out as rose or violet spots (Fig S5) A con- centration of 0.1 g L −1 resazurin was chosen as final concentration for the spraying because this resulted in a complete conversion of the resazurin to the colorless dihydroresorufin and thus provided the highest contrast to the background Second, cytotoxic signals
of different amounts of 4-NQO and BPA were scanned at five dif- ferent wavelengths ( λ = 350 nm, 360 nm, 520 nm, 540 and 575 nm) (Fig S6) A wavelength of 575 nm resulted in the highest sig-
Trang 5Fig 2 Comparison of planar yeast androgen screen (p-YAS, left) and planar yeast anti-androgen screen (p-YAAS, right) shown with the model compounds testosterone and
flutamide applied as a mix (3.33 μg L −1 testosterone and 5 mg L −1 flutamide) or individually A two-step chromatographic development was performed using 1.) methanol and 2.) ethyl acetate:n-hexane 50:50 (V:V) The image shows the signal detection with fluorescence-imaging at λexcitation = 366 nm without (left) and with (right) testosterone spike (50 ng mL −1 )
nal intensity and was thus selected as final measuring wavelength
Finally, different incubation times after resazurin application were
investigated (t = 5 min, 15 min, 30 min and 60 min) (Fig S7) An
incubation time of 30 min resulted in the clearest signals and the
best contrast to the background and was therefore chosen as final
incubation time
The distinction between anti-androgenic and cytotoxic effects
on the same HPTLC plate was tested using this optimized proce- dure Therefore, the anti-androgenic model compounds BPA and flutamide and the cytotoxic 4-NQO were applied in different amounts on a HPTLC plate First, the p-YAAS was performed to de- tect anti-androgenic activity ( Fig 3a.)) After signal detection, re-
Fig 3 Distinction between cytotoxic and anti-androgenic effects using the planar yeast anti-androgen screen (p-YAAS) in combination with a resazurin cell viability assay
without chromatographic separation The model compounds bisphenol A (anti-androgenic), flutamide (anti-androgenic) and 4-nitroquinoline 1-oxide (cytotoxic) were applied
on a HPTLC plate in amounts indicated above the images The two images show the same HPTLC plate: a.) Anti-androgenic effect detection by testosterone spike (50 ng
mL −1 ) with fluorescence-imaging at λexcitation = 366 nm, b.) cytotoxic effect detection with resazurin under white light
Trang 6Fig 4 Analysis of a.) anti-androgenic and b.) cytotoxic effects in different dilutions of a landfill leachate extract using the planar yeast anti-androgen screen (p-YAAS) in
combination with a resazurin cell viability assay a.) fluorescence-imaging at λexcitation = 366 nm, b.) signal detection under white light Landfill leachate extracts were applied
in different volumes on a HPTLC plate as indicated at the top of the figure and subsequently separated in a two-step chromatographic development with 1.) methanol and 2.) chloroform:EtAc:petroleum fraction (55:20:25, V:V:V, modified after Cimpoiu, et al [31] ) 4-Nitroquinoline 1-oxide (4-NQO), bisphenol A (BPA) and flutamide served as positive controls applied on the three rightmost tracks
sazurin was sprayed on the same HPTLC plate to visualize possible
cytotoxic effects ( Fig 3b.)) Based on the results shown in Fig 3,
the signal suppression of the p-YAAS caused by 4-NQO can be at-
tributed clearly to a cytotoxic effect whereas the absence of cyto-
toxic effects indicates the specific antagonistic effects of BPA and
flutamide in the amounts applied to the HPTLC plate
3.4 Analysis of antagonistic and cytotoxic effects in a landfill
leachate extract
Finally, the applicability of the developed method for envi-
ronmental samples was investigated Environmental samples often
contain complex matrices composed of several compound classes
which potentially can affect the performance of bioassays For a
proof of concept, the p-YAAS and the planar cytotoxicity assay
were applied to an extracted landfill leachate Despite the over-
load of the stationary phase as evident by insufficiently sepa-
rated and elongated and/or broadened signals, the application of
the undiluted sample extract led to the detection of three anti-
androgenic spots Two of this three anti-androgenic fractions ad-
ditionally showed cytotoxic effects as demonstrated with the re-
sazurin cell viability assay performed directly after the p-YAAS
on the same HPTLC plate ( Fig 4b.)) These areas of cytotoxicity are located in the center of the respective signals detected by the p-YAAS After dilution of the sample, anti-androgenic effects were detected in each sample dilution as two separated signals of comparable intensity and with dose-dependent variation of inten- sity ( Fig.4a.)) The upper signals shared the same Rf-value as the model compound BPA In contrast, the lower signals could not be assigned to a candidate compound The androgenic activity of the same sample had been investigated by p-YAS in the course of an earlier study conducted by Riegraf, et al [32] In contrast to the anti-androgenic and cytotoxic effects, androgenicity was not ob- served in the tested concentrations
For the quantification of antagonistic effects, the diluted sample extract was investigated in three different volumes on the HPTLC plate in parallel to a calibration spanning the amounts of 10 to
500 ng BPA and flutamide (Fig S8) The equivalent concentrations were calculated as arithmetic mean of three independent exper- iments resulting in a total of nine replicates The upper signal corresponded to 0.45 ± 0.08 mg BPA-EQ L −1 or 1.26 ± 0.19 mg flutamide-EQ L −1in the original leachate Furthermore, 1.10 ± 0.20
6
Trang 7mg flutamide-EQ L −1 were calculated for the lower signal of anti-
androgenicity In total, 2.36 ± 0.38 mg flutamide-EQ L −1 were de-
termined in the landfill leachate under the assumption of a quan-
titative extraction of the compounds
4 Discussion
In the presented study, a procedure based on the direct cou-
pling of normal phase HPTLC with the yeast anti-androgen screen
for the detection of anti-androgenic effects was successfully estab-
lished using the model compounds flutamide and BPA The de-
tectability of specific antagonistic effects on the androgen-receptor
in contrast to general inhibitory effects such as cytotoxicity is
clearly underlined by the observation that the antagonistic ef-
fects mediated by flutamide and BPA can be masked by increasing
spike-levels of testosterone (Fig S1) In contrast, the signal sup-
pression of the fluorescent background by the cytotoxic 4-NQO was
not affected by the spike-level of the receptor agonist (Fig S2) in-
dicating that the inhibitory effect is not mediated by the andro-
gen receptor but by its toxicity to the yeast cells In general, all ef-
fects of compounds reducing the activity of the androgen receptor,
e.g., via a competition for the ligand binding domain or allosteric
regulation would be detectable by the proposed assay Itzel, et al
[44]provides an overview of 89 compounds whose anti-androgenic
action was verified in different bioassays However, as for all cell-
based in vitro-assays the definite outcome depends on the cellular
context and modes of action For example, interferences with the
binding of the androgen receptor to individual, cell-specific tran-
scriptional cofactors might escape the detection by the proposed
assay [ 45, 46]
The relative potency of BPA to flutamide was found to be
0.68 without and 1.47 with chromatographic development Sim-
ilar results were reported by Rostkowski, et al [10] who deter-
mined a relative potency for BPA of 0.60 by a YAAS in microtiter
plate format These findings also correlate well with that of Fang,
et al [47] who detected comparable binding affinities for flu-
tamide and BPA to the androgen receptor The shift in the rela-
tive potency caused by the chromatographic development in the
proposed method might be explained by a different diffusion of
BPA compared to flutamide during the development of the HPTLC
plate which lead to small shifts in the dose response relationship
The resulting changes of relative potencies have to be considered,
e.g., for the calculation of effect contributions of compounds in a
mixture-based on data of chemical analysis
Due to the possibility to apply high sample volumes to the
HPTLC plate, the presented method has a higher effective sensitiv-
ity compared to its equivalent in the 96-well format Assuming a
common 10 0 0-fold enrichment of environmental samples, the de-
termined ED10 of 27.9 ng flutamide per spot translates to a LOD
of 5.58 μg L −1(0.02 μM) in case of an application volume of 5 μL
and even only 0.28 μg L −1(0.001 μM) after the application of 100
μL sample extract In comparison, the inhibitory concentration as-
sociated with 10% and 50% effect (IC10 and IC50) of flutamide us-
ing the same yeast strain in the classic 96-well plate approach was
1.53 ± 0.19 mg L −1(5.56 μM) and 4.29 ± 0.32 mg L −1 (15.54 μM),
respectively (Fig S4) These results are in line with the sensitivity
of other recombinant yeast strains expressing the human andro-
gen receptor, which e g obtained an IC50 of 6.14 μM or 20.3 μM
of flutamide in microtiter plates [ 48, 49] Assays based on mam-
malian cell lines are considered to detect androgen receptor medi-
ated effects more sensitive than yeast-based assays [50] For exam-
ple, Hu, et al [51]recently reported an IC50 of 2.3 μM flutamide
using an assay based on MDA-kb2 cells In the context of an inter-
national ring trial, the IC50 detected by the anti-AR-CALUX method
actually ranged between 0.11 and 1.1 μM flutamide [52] Since the
experimental set-up of the study published by Klingelhöfer, et al
[31]using reverse-phase HPTLC plates was not designed for quan- tification purposes, a direct comparison of sensitivities is not pos- sible Only the higher spike-level of testosterone in the RP-HPTLC (800 pg testosterone/mm −2 instead of 7.5 pg testosterone/mm −2) might indicate a higher sensitivity of the assay based on normal phase HPTLC The proposed method allows the detection of anti- androgenicity in the range of suggested effect-based trigger val- ues of 3.3 to 14.4 μg flutamide-EQ L −1 [53] When compared with chemical target analyses for flutamide, the ED10 of the method [52]presented is in the same order of magnitude as the LOD of an approach using HPLC-UV [54]and even lower than that of an elec- trochemical sensor recently developed for the trace-level recogni- tion in biofluids [55]
By the application of the p-YAAS (and p-YAS), androgenic and anti-androgenic compounds can be spatially separated prior to the application of the test organisms ( Fig.2) This is an advantage for the investigation of complex environmental samples For example, Pannekens, et al [56] showed that counter-acting substances, i.e receptor agonists and antagonists, concurrently occur in wastewa- ters from municipal and hospital WWTPs resulting in suppressed biological signals in reporter gene assays Agonist effects can even
be completely masked by antagonistic compounds as shown by Weiss, et al [11]for androgenic effects in river sediments Sample fractionation could be done in a higher resolution by HPLC, how- ever, an increased sample throughput would require investments
in lab-automation Furthermore, used mobile phases for separa- tion in HPLC might interfere with subsequent bioassays if not com- pletely removed by evaporation [57] The proposed method allows
a simultaneous analysis of ten samples per HPTLC plate without the need to prepare and test dilution series of the sample, due to the inherent dilution of the sample on the surface of the HPTLC plate by diffusion processes Thus, specific anti-androgenic effects are detectable even at high sample concentrations leading to cy- totoxic effects as shown for the undiluted extract of the landfill leachate The developed planar cytotoxicity assay was performed
on the same HPTLC plate subsequent to the p-YAAS reducing cost and time-need considerably Cytotoxic effects caused by the ex- tracted landfill leachate were detectable unambiguously as colored spots ( Fig 4b) specific anti-androgenic effects are detectable in parallel in the areas surrounding the cytotoxic center of the signal ( Fig.4a)
The total concentration of anti-androgenic substances in the landfill leachate equals to 2.36 ± 0.38 mg flutamide-EQ L −1 and vastly exceeded the levels of 11.7 to 56.4 μg flutamide-EQ L −1 found in municipal and hospital wastewaters [56] Furthermore, 6
to 32 μg flutamide-EQ L −1were reported in the context of investi- gations on effluents of 12 WWTPs in Danube river basin [58]and levels of up to 90 μg flutamide-EQ L −1 were detected during a two-year survey at three Dutch surface waters [59] Thus, the anti- androgenic effect in the landfill leachate was around 25 to 400 times higher than that previously reported in waste- and river wa- ters Escher, et al [53]suggested an effect-based trigger value for anti-androgenicity in the range of 3.3 to 14.4 μg flutamide-EQ L −1 This threshold was exceeded by a factor of about 160 to 700 by the landfill leachate However, the investigated leachate is not a di- rect threat to the environment since it is treated onsite before it is discharged into a municipal WWTP for further treatment Though
a leakage of the draining system might lead to a contamination
of surrounding soils and water bodies with anti-androgenic com- pounds
The calculated BPA-EQ of 0.45 ± 0.08 mg L −1for the upper an- tagonistic signal with the same Rf-value as the model compound BPA is in the same range as the previously reporter BPA concen- tration of 2.9 mg L −1 determined by GC-MS/MS [32] This study of Riegraf, et al additionally revealed the presence of nonylphenols and 4- tert-octylphenol in the respective landfill leachate Although
Trang 8both substances showed anti-androgenic activities in prior reporter
gene assays [ 10, 60], the observed antagonistic effects cannot be
assigned to nonylphenols and 4- tert-octylphenol as their retarda-
tion factor did not correspond to the main signals detected in the
sample However, the third anti-androgenic signal only detectable
in the undiluted sample extract could be caused by nonylphenols
and 4- tert-octylphenol as they show a similar migration distance
under identical separation conditions [32] In contrast to BPA, these
two compounds have not been quantified by chemical analysis, so
that their expected anti-androgenic effect cannot be estimated As
the complete explanation of anti-androgenic effects in the landfill
leachate was out of the scope of our study, the identification of
causing compounds was not pursued any further The extraction
of the stationary phase at relevant positions and the subsequent
analysis by mass-spectrometry could support the identification of
bioactive compounds in terms of an effect-directed analysis as pro-
posed by Weiss, et al [61]
5 Conclusion
Coupling HPTLC with bioassays to detect anti-androgenic ac-
tivity and cytotoxicity in parallel allows a matrix-robust, cost-
efficient, fast and sensitive elucidation of effects and a reduction of
interferences from agonists of the androgen receptor as well as cy-
totoxic effects that might lead to false positive test results The p-
YAAS allows the detection of a group of toxic substances with high
environmental relevance and is thus a valuable addition to the ex-
isting methods that combine HPTLC and various specific effect-
based bioassays The proposed method helps to reduce the num-
ber of dilution steps, e.g., needed in liquid culture assays, while at
the same time increasing the possibility to detect very low concen-
trations of compounds of interest offering a wide range of possible
applications in environmental monitoring So far, only a limited set
of model compounds and sample extracts was investigated in the
present study The routine investigation of anti-androgenic effects
using the proposed method can improve the correlation between
results of chemical analyses and the overall anti-androgenic activ-
ity, because an underestimation of effects due to a mutual masking
of agonistic and antagonistic compounds but as well false positive
test results can be avoided
Data availability statement
The data that support the findings of this study are available
from the corresponding author upon reasonable request
Declaration of competing interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper
CRediT authorship contribution statement
Carolin Riegraf: Conceptualization, Formal analysis, Validation,
Visualization, Writing – original draft Anna Maria Bell: Formal
analysis, Validation, Visualization, Writing – original draft Marina
Ohlig: Data curation, Investigation, Writing – review & edit-
ing Georg Reifferscheid: Writing – review & editing Sebastian
Buchinger: Conceptualization, Supervision, Writing – review &
editing
Data availability
Data will be made available on request
Acknowledgement
This work was supported by the German Federal Ministry for the Environment, Nature Conservation, Nuclear Safety and Con- sumer Protection in line of the BMUV-project ‘General and specific ecotoxicology’ The authors thank Ramona Pfänder for the excel- lent technical assistance with the yeast assays in microtiter plate format
Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.chroma.2022.463582
References
[1] A Pal, K.Y.-H Gin, A.Y.-C Lin, M Reinhard, Impacts of emerging organic con- taminants on freshwater resources: review of recent occurrences, sources, fate and effects, Sci Total Environ 408 (2010) 6062–6069, doi: 10.1016/j.scitotenv 2010.09.026
[2] D.J Lapworth, N Baran, M.E Stuart, R.S Ward, Emerging organic contaminants
in groundwater: a review of sources, fate and occurrence, Environ Pollut 163 (2012) 287–303, doi: 10.1016/j.envpol.2011.12.034
[3] S Jobling, N Beresford, M Nolan, T Rodgers-Gray, G.C Brighty, J.P Sumpter, C.R Tyler, Altered sexual maturation and gamete production in wild roach (Rutilus rutilus) living in rivers that receive treated sewage effluents1, Biol Re- prod 66 (2002) 272–281, doi: 10.1095/biolreprod66.2.272
[4] K.A Kidd, M.J Paterson, M.D Rennie, C.L Podemski, D.L Findlay, P.J Blanch- field, K Liber, Direct and indirect responses of a freshwater food web to
a potent synthetic oestrogen, Philos Trans R Soc B 369 (2014) 20130578, doi: 10.1098/rstb.2013.0578
[5] R.J Ellis, M.R van den Heuvel, E Bandelj, M.A Smith, L.H McCarthy, T.R Stuthridge, D.R Dietrich, In vivo and in vitro assessment of the androgenic potential of a pulp and paper mill effluent, Environ Toxicol Chem 22 (2003) 1448–1456, doi: 10.1002/etc.5620220705
[6] E.P Kolodziej, D.L Sedlak, Rangeland grazing as a source of steroid hormones
to surface waters, Environ Sci Technol 41 (2007) 3514–3520, doi: 10.1021/ es063050y
[7] J Durhan Elizabeth, S Lambright Christy, A Makynen Elizabeth, J Lazorchak,
C Hartig Phillip, S Wilson Vickie, L.E Gray, T Ankley Gerald, Identification of metabolites of Trenbolone acetate in androgenic runoff from a beef feedlot, Environ Health Perspect 114 (2006) 65–68, doi: 10.1289/ehp.8055
[8] V Kumar, C Majumdar, P Roy, Effects of endocrine disrupting chemicals from leather industry effluents on male reproductive system, J Steroid Biochem Mol Biol 111 (2008) 208–216, doi: 10.1016/j.jsbmb.20 08.06.0 05
[9] E.M Hill, K.L Evans, J Horwood, P Rostkowski, F.O Oladapo, R Gib- son, J.A Shears, C.R Tyler, Profiles and some initial identifications of (Anti)Androgenic compounds in fish exposed to wastewater treatment works effluents, Environ Sci Technol 44 (2010) 1137–1143, doi: 10.1021/es901837n [10] P Rostkowski, J Horwood, J.A Shears, A Lange, F.O Oladapo, H.T Besselink, C.R Tyler, E.M Hill, Bioassay-directed identification of novel antiandrogenic compounds in bile of fish exposed to wastewater effluents, Environ Sci Tech- nol 45 (2011) 10660–10667, doi: 10.1021/es202966c
[11] J.M Weiss, T Hamers, K.V Thomas, S van der Linden, P.E.G Leonards, M.H Lamoree, Masking effect of anti-androgens on androgenic activity in Eu- ropean river sediment unveiled by effect-directed analysis, Anal Bioanal.Chem
394 (2009) 1385–1397, doi: 10.1007/s00216- 009- 2807- 8 [12] S Milla, S Depiereux, P Kestemont, The effects of estrogenic and androgenic endocrine disruptors on the immune system of fish: a review, Ecotoxicol 20 (2011) 305–319, doi: 10.1007/s10646-010- 0588- 7
[13] Y Kiparissis, T.L Metcalfe, G.C Balch, C.D Metcalfe, Effects of the antian- drogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes), Aquat Toxicol 63 (2003) 391–403, doi: 10 1016/S0166-445X(02)00189-3
[14] L Gehrmann, H Bielak, M Behr, F Itzel, S Lyko, A Simon, G Kunze, E Dopp,
M Wagner, J Tuerk, Anti-)estrogenic and (anti-)androgenic effects in wastew- ater during advanced treatment: comparison of three in vitro bioassays, Envi- ron Sci Pollut Res Int 25 (2018) 4094–4104, doi: 10.1007/s11356- 016- 7165- 4 [15] T.J Runnalls, L Margiotta-Casaluci, S Kugathas, J.P Sumpter, Pharma- ceuticals in the aquatic environment: steroids and anti-steroids as high priorities for research, Hum Ecol Risk Assess., 16 (2010) 1318-1338 https://doi.org/10.1080/10807039.2010.526503
[16] J.M Weiss, E Simon, G.J Stroomberg, R de Boer, J de Boer, S.C van der Lin- den, P.E.G Leonards, M.H Lamoree, Identification strategy for unknown pol- lutants using high-resolution mass spectrometry: androgen-disrupting com- pounds identified through effect-directed analysis, Anal Bioanal.Chem 400 (2011) 3141–3149, doi: 10.10 07/s0 0216- 011- 4939- x
[17] P Sohoni, J Sumpter, Several environmental oestrogens are also anti- androgens, J Endocrinol 158 (1998) 327, doi: 10.1677/joe.0.1580327 [18] K.V Thomas, K Langford, K Petersen, A.J Smith, K.E Tollefsen, Effect-directed identification of naphthenic acids as important in Vitro Xeno-estrogens and
8
Trang 9anti-androgens in north sea offshore produced water discharges, Environ Sci
Technol 43 (2009) 8066–8071, doi: 10.1021/es9014212
[19] M Muschket, C Di Paolo, A.J Tindall, G Touak, A Phan, M Krauss, K Kirch-
ner, T.-B Seiler, H Hollert, W Brack, Identification of unknown antiandro-
genic compounds in surface waters by effect-directed analysis (EDA) using
a parallel fractionation approach, Environ Sci Technol 52 (2018) 288–297,
doi: 10.1021/acs.est.7b04994
[20] M.L Eldridge, J Sanseverino, A.C Layton, J.P Easter, T.W Schultz, G.S Sayler,
Saccharomyces cerevisiae BLYAS, a new Bioluminescent bioreporter for detec-
tion of androgenic compounds, Appl Environ Microbiol 73 (2007) 6012–6018,
doi: 10.1128/aem.00589-07
[21] B van der Burg, R Winter, H.-y Man, C Vangenechten, P Berckmans,
M Weimer, H Witters, S van der Linden, Optimization and prevalidation of
the in vitro AR CALUX method to test androgenic and antiandrogenic activity
of compounds, Reprod Toxicol 30 (2010) 18–24, doi: 10.1016/j.reprotox.2010
04.012
[22] V.S Wilson, K Bobseine, C.R Lambright, L.E Gray 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 66
(2002) 69–81, doi: 10.1093/toxsci/66.1.69
[23] C.J Houtman, R ten Broek, A Brouwer, Steroid hormonal bioactivities, cul-
prit natural and synthetic hormones and other emerging contaminants in
waste water measured using bioassays and UPLC-tQ-MS, Sci Total Environ 630
(2018) 1492–1501, doi: 10.1016/j.scitotenv.2018.02.273
[24] A Abbas, I Schneider, A Bollmann, J Funke, J Oehlmann, C Prasse, U Schulte-
Oehlmann, W Seitz, T Ternes, M Weber, H Wesely, M Wagner, What you
extract is what you see: Optimising the preparation of water and wastewa-
ter samples for in vitro bioassays, Water Res 152 (2019) 47–60, doi: 10.1016/j
watres.2018.12.049
[25] M.A.K Hashmi, M Krauss, B.I Escher, I Teodorovic, W Brack, Effect-directed
analysis of progestogens and glucocorticoids at trace concentrations in river
water, Environ Toxicol Chem 39 (2020) 189–199, doi: 10.1002/etc.4609
[26] G Morlock, W Schwack, Hyphenations in planar chromatography, J Chro-
matogr A 1217 (2010) 6600–6609, doi: 10.1016/j.chroma.2010.04.058
[27] S Buchinger, D Spira, K Bröder, M Schlüsener, T Ternes, G Reifferscheid, Di-
rect coupling of thin-layer chromatography with a bioassay for the detection
of estrogenic compounds: applications for effect-directed analysis, Anal Chem
85 (2013) 7248–7256, doi: 10.1021/ac4010925
[28] A.J Bergmann, E Simon, A Schifferli, A Schönborn, E.L.M Vermeirssen, Es-
trogenic activity of food contact materials—evaluation of 20 chemicals using
a yeast estrogen screen on HPTLC or 96-well plates, Anal Bioanal.Chem 412
(2020) 4527–4536, doi: 10.10 07/s0 0216- 020- 02701- w
[29] C Riegraf, G Reifferscheid, S Belkin, L Moscovici, D Shakibai, H Hollert,
S Buchinger, Combination of yeast-based in vitro screens with high-
performance thin-layer chromatography as a novel tool for the detection of
hormonal and dioxin-like compounds, Anal Chim Acta 1081 (2019) 218–230,
doi: 10.1016/j.aca.2019.07.018
[30] C Riegraf, G Reifferscheid, B Becker, S Belkin, H Hollert, U Feiler,
S Buchinger, Detection and quantification of photosystem II inhibitors us-
ing the freshwater Alga Desmodesmus subspicatus in combination with
high-performance thin-layer chromatography, Environ Sci Technol 53 (2019)
13458–13467, doi: 10.1021/acs.est.9b04634
[31] I Klingelhöfer, N Hockamp, G.E Morlock, Non-targeted detection and differen-
tiation of agonists versus antagonists, directly in bioprofiles of everyday prod-
ucts, Anal Chim Acta 1125 (2020) 288–298, doi: 10.1016/j.aca.2020.05.057
[32] C Riegraf, G Reifferscheid, L Moscovici, D Shakibai, H Hollert, S Belkin,
S Buchinger, Coupling high-performance thin-layer chromatography with a
battery of cell-based assays reveals bioactive components in wastewater and
landfill leachates, Ecotoxicol Environ Saf 214 (2021) 112092, doi: 10.1016/j
ecoenv.2021.112092
[33] A Schoenborn, P Schmid, S Bräm, G Reifferscheid, M Ohlig, S Buchinger, Un-
precedented sensitivity of the planar yeast estrogen screen by using a spray-on
technology, J Chromatogr A 1530 (2017) 185–191, doi: 10.1016/j.chroma.2017
11.009
[34] C Cimpoiu, A Hosu, S Hodisan, Analysis of some steroids by thin-layer chro-
matography using optimum mobile phases, J Pharm Biomed Anal 41 (2006)
633–637, doi: 10.1016/j.jpba.20 05.12.0 04
[35] I.J Purvis, D Chotai, C.W Dykes, D.B Lubahn, F.S French, E.M Wilson,
A.N Hobden, An androgen-inducible expression system for Saccharomyces
cerevisiae, Gene 106 (1991) 35–42, doi: 10.1016/0378-1119(91)90563-Q
[36] ISO 7027-1, International organization for standardization Water quality — De-
termination of turbidity — Part 1: quantitative methods., in, 2016
[37] R Development Core TeamR: A Language and Environment for Statistical Com-
puting, R Foundation for Statistical Computing, Vienna, Austria, 2008 http:
//www.R-project.org
[38] C Ritz, F Baty, J.C Streibig, D Gerhard, Dose-response analysis using R, PLoS
One 10 (2016) e0146021, doi: 10.1371/journal.pone.0146021
[39] H Wickham, ggplot2: Elegant Graphics for Data Analysis, Springer, New York,
2016, doi: 10.1007/978- 3- 319- 24277- 4
[40] C Ritz, Toward a unified approach to dose–response modeling in ecotoxicol-
ogy, Environ Toxicol Chem 29 (2010) 220–229, doi: 10.1002/etc.7
[41] M Jaskiewicz, M Orlowska, G Olizarowicz, D Migon, D Grzywacz, W Kamysz, Rapid screening of antimicrobial synthetic peptides, Int J Pept Res Ther 22 (2016) 155–161, doi: 10.1007/s10989-015-9494-4
[42] H Dehghan, P Rezaee, A Aliahmadi, Bioassay screening of 12 Iranian plants and detection of antibacterial compounds from Heracleum persicum using a TLC bioautography method, J Liq Chromatogr Relat Technol 43 (2020) 381–
387, doi: 10.1080/10826076.2020.1725557 [43] I Klingelhofer, L.P Ngoc, B van der Burg, G.E Morlock, A bioimaging system combining human cultured reporter cells and planar chromatography to iden- tify novel bioactive molecules, Anal Chim Acta 1183 (2021) 11, doi: 10.1016/j aca.2021.338956
[44] F Itzel, L Gehrmann, T Teutenberg, T.C Schmidt, J Tuerk, Recent develop- ments and concepts of effect-based methods for the detection of endocrine activity and the importance of antagonistic effects, TrAC, Trends Anal Chem
118 (2019) 699–708, doi: 10.1016/j.trac.2019.06.030 [45] Y Hikichi, M Yamaoka, M Kusaka, T Hara, Selective androgen receptor modu- lator activity of a steroidal antiandrogen TSAA-291 and its cofactor recruitment profile, Eur J Pharmacol 765 (2015) 322–331, doi: 10.1016/j.ejphar.2015.08.052 [46] H Dotzlaw, U Moehren, S Mink, A.C.B Cato, J.A.I Lluhi, A Baniahmad, The amino terminus of the human AR is target for corepressor action and antihor- mone agonism, Mol Endocrinol 16 (2002) 661–673, doi: 10.1210/me.16.4.661 [47] H Fang, W.D Tong, W.S Branham, C.L Moland, S.L Dial, H.X Hong, Q Xie,
R Perkins, W Owens, D.M Sheehan, Study of 202 natural, synthetic, and envi- ronmental chemicals for binding to the androgen receptor, Chem Res Toxicol
16 (2003) 1338–1358, doi: 10.1021/tx030011g [48] J Stragierowicz, S Stypula-Trebas, L Radko, A Posyniak, M Nasiadek, M Klim- czak, A Kilanowicz, An assessment of the estrogenic and androgenic properties
of tetra- and hexachloronaphthalene by YES/YAS in vitro assays, Chemosphere
263 (2021) 10, doi: 10.1016/j.chemosphere.2020.128006 [49] D.H Kim, C.G Park, S.H Kim, Y.J Kim, The Effects of Mono-(2-Ethylhexyl) Phthalate (MEHP) on human estrogen receptor (hER) and androgen recep- tor (hAR) by YES/YAS In Vitro assay, Molecules 24 (2019) 10, doi: 10.3390/ molecules24081558
[50] G Bagchi Bhattacharjee, S.M Paul Khurana, In vitro reporter assays for screen- ing of chemicals that disrupt androgen signaling, J Toxicol 2014 (2014)
701752, doi: 10.1155/2014/701752 [51] X.X Hu, W Shi, S Wei, X.W Zhang, H.X Yu, Identification of (anti-)androgenic activities and risks of sludges from industrial and domestic wastewater treat- ment plants, Environ Pollut 268 (2021) 9, doi: 10.1016/j.envpol.2020.115716 [52] A Milcamps, R Liska, I Langezaal, W Casey, M Dent, J Odum, Reliabil- ity of the AR-CALUX (R) In Vitro method used to detect chemicals with (Anti)Androgen activity: results of an international ring trial, Toxicol Sci 184 (2021) 170–182, doi: 10.1093/toxsci/kfab078
[53] B.I Escher, S A ї t-A ї ssa, P.A Behnisch, W Brack, F Brion, A Brouwer,
S Buchinger, S.E Crawford, D Du Pasquier, T.J.S.o.t.T.E Hamers, Effect- based trigger values for in vitro and in vivo bioassays performed on surface water extracts supporting the environmental quality standards (EQS) of the European water framework directive, 628 (2018) 748-765 https://doi.org/10.1016/j.scitotenv.2018.01.340
[54] H Bhatia, A Kumar, Y Ogino, J Du, A Gregg, J Chapman, M.J McLaughlin,
T Iguchi, Effects of the commercial antiandrogen flutamide on the biomarkers
of reproduction in male Murray rainbowfish (Melanotaenia fluviatilis), Environ Toxicol Chem 33 (2014) 1098–1107, doi: 10.1002/etc.2524
[55] L Durai, A Gopalakrishnan, S Badhulika, A low-cost and facile electrochemical sensor for the trace-level recognition of flutamide in biofluids using large-area bimetallic NiCo2O4 micro flowers, New J Chem 46 (2022) 3383–3391, doi: 10 1039/d1nj05246b
[56] H Pannekens, A Gottschlich, H Hollert, E Dopp, Evaluation of mixture effects
of endocrine active substances in wastewater using CALUX reporter-gene as- says, Int J Hyg Environ Health 222 (2019) 670–677, doi: 10.1016/j.ijheh.2019 04.008
[57] M.G Weller, A unifying review of bioassay-guided fractionation, effect-directed analysis and related techniques, Sensors 12 (2012) 9181–9209, doi: 10.3390/ s120709181
[58] N.A Alygizakis, H Besselink, G.K Paulus, P Oswald, L.M Hornstra, M Oswal- dova, G Medema, N.S Thomaidis, P.A Behnisch, J Slobodnik, Characterization
of wastewater effluents in the Danube River Basin with chemical screening, in vitro bioassays and antibiotic resistant genes analysis, Environ Int 127 (2019) 420–429, doi: 10.1016/j.envint.2019.03.060
[59] C.J Houtman, K Brewster, R ten Broek, B Duijve, Y van Oorschot, M Rosielle, M.H Lamoree, R Steen, Characterisation of (anti-)progestogenic and (anti- )androgenic activities in surface and wastewater using high resolution effect- directed analysis, Environ Int 153 (2021) 13, doi: 10.1016/j.envint.2021.106536 [60] J Li, M Ma, Z.J Wang, In vitro profiling of endocrine disrupting effects of phe- nols, Toxicol Vitro 24 (2010) 201–207, doi: 10.1016/j.tiv.20 09.09.0 08 [61] S.C Weiss, N Egetenmeyer, W Schulz, Coupling of in vitro bioassays with planar chromatography in effect-directed analysis, in: G Reifferscheid,
S Buchinger (Eds.), Vitro Environmental Toxicology - Concepts, Application and Assessment, Springer International Publishing Ag, Cham, 2017, pp 187–224, doi: 10.1007/10 _ 2016 _ 16