Disruptive effects of plasticizers bisphenol A, F, and S on steroidogenesis of adrenocortical cells

Introduction Endocrine disrupting chemicals (EDCs) are known to interfere with endocrine homeostasis. Their impact on the adrenal cortex and steroidogenesis has not yet been sufficiently elucidated. This applies in particular to the ubiquitously available bisphenols A (BPA), F (BPF), and S (BPS). Methods NCI-H295R adrenocortical cells were exposed to different concentrations (1nM-1mM) of BPA, BPF, BPS, and an equimolar mixture of them (BPmix). After 72 hours, 15 endogenous steroids were measured using LC-MS/MS. Ratios of substrate and product of CYP-regulated steps were calculated to identify most influenced steps of steroidogenesis. mRNA expression of steroidogenic enzymes was determined by real-time PCR. Results Cell viability remained unaffected at bisphenol concentrations lower than 250 µM. All tested bisphenols and their combination led to extensive alterations in the quantified steroid levels. The most profound fold changes (FC) in steroid concentrations after exposure to BPA (>10µM) were seen for androstenedione, e.g. a 0.37±0.11-fold decrease at 25µM (p≤0.0001) compared to vehicle-treated controls. For BPF, levels of 17-hydroxyprogesterone were significantly increased by 25µM (FC 2.57±0.49, p≤0.001) and 50µM (FC 2.65±0.61, p≤0.0001). BPS treatment led to a dose-dependent decrease of 11-deoxycorticosterone at >1µM (e.g. FC 0.24±0.14, p≤0.0001 at 10µM). However, when combining all three bisphenols, additive effects were detected: e.g. 11-deoxycortisosterone was decreased at doses >10µM (FC 0.27±0.04, p≤0.0001, at 25µM), whereas 21-deoxycortisol was increased by 2.92±0.20 (p≤0.01) at 10µM, and by 3.21±0.45 (p≤0.001) at 50µM. While every measured androgen (DHEA, DHEAS, androstenedione, testosterone, DHT) was lowered in all experiments, estradiol levels were significantly increased by BPA, BPF, BPS, and BPmix (e.g. FC 3.60±0.54, p≤0.0001 at 100µM BPF). Calculated substrate-product ratios indicated an inhibition of CYP17A1-, and CYP21A2 mediated conversions, whereas CYP11B1 and CYP19A1 showed higher activity in the presence of bisphenols. Based on these findings, most relevant mRNA expression of CYP genes were analysed. mRNA levels of StAR, CYP11B1, and CYP17A1 were significantly increased by BPF, BPS, and BPmix. Discussion In cell culture, bisphenols interfere with steroidogenesis at non-cytotoxic levels, leading to compound-specific patterns of significantly altered hormone levels. These results justify and call for additional in-vivo studies to evaluate effects of EDCs on adrenal gland functionality.

Introduction: Endocrine disrupting chemicals (EDCs) are known to interfere with endocrine homeostasis.Their impact on the adrenal cortex and steroidogenesis has not yet been sufficiently elucidated.This applies in particular to the ubiquitously available bisphenols A (BPA), F (BPF), and S (BPS).
Methods: NCI-H295R adrenocortical cells were exposed to different concentrations (1nM-1mM) of BPA, BPF, BPS, and an equimolar mixture of them (BPmix).After 72 hours, 15 endogenous steroids were measured using LC-MS/MS.Ratios of substrate and product of CYP-regulated steps were calculated to identify most influenced steps of steroidogenesis.mRNA expression of steroidogenic enzymes was determined by real-time PCR.
Discussion: In cell culture, bisphenols interfere with steroidogenesis at noncytotoxic levels, leading to compound-specific patterns of significantly altered hormone levels.These results justify and call for additional in-vivo studies to evaluate effects of EDCs on adrenal gland functionality.

Introduction
Endocrine disrupting chemicals (EDC) are substances, which interfere with endocrine homeostasis and therefore promote adverse health outcomes (1).Although the evidence for their harmful effects and hazardous potential is growing constantly, there is little regulation on how the disruptive potential of a new substance must be assessed before it is released (2).
Generally, there are numerous chemicals, with which man comes into contact on a daily base, while knowledge is limited about their disrupting potential.EDCs can be found in drinking water and food even years after they have been banned in one region of the world.They can be detected in various human samples like seminal plasma (3), adipose tissue (4), or even amniotic fluid (5).Prominent examples for such EDCs are bisphenol A (BPA) or the structurally similar substances bisphenol F (BPF) and S (BPS), which are nowadays regularly used as substitutes for BPA.
BPA, F and S are aromatic, organic compounds related to diphenylmethane.Used as additive plasticizers in polycarbonate plastics and as primary material for epoxy resins, bisphenols appear in a broad range of applications.They are components of food containers, coatings of food cans, plastic bottles, thermal paper or medical products.In 2021 alone, more than 6 million tons of BPA were produced and distributed worldwide (6).However, not chemically bound to the polymer structure, bisphenols can leach out of the compound, generally promoted by heat, radiation, or weathering processes.Ubiquitously detectable micro-and nanoplastics show high fractions of bisphenols and other EDCs (7).EU-wide biomonitoring was able to detect bisphenols in over 92% of analyzed samples (8).Assuming that the general population is regularly exposed to nanomolar doses, maximum bisphenol levels of 8.48μM (9) or 6.85μM (10) have been measured in occupationally exposed workers.The European Food Safety Authority (EFSA) recently corrected their recommendation for the tolerated intake dose (TDI) for BPA from 4μg/kg body weight/day downwards to 0.2 ng/kg body weight/day following the many indications of risks for public health from the presence of BPA especially in foodstuffs.(7).However, the substitutes BPF and S are still not strictly regulated.
As part of the registration procedure of the OECD (Organization for Economic Co-operation and Development) for substances before approval for distribution, the adrenocortical carcinoma-derived cell line NCI-H295R is used to detect alterations in the secretion by two representative steroids, testosterone and estradiol (11,12).but further clinically relevant hormones, such as mineralo-, glucocorticoids or adrenal androgens are not taken into account.
Contrary to their effects on the thyroid gland (13,14), the effects of EDCs on the adrenal gland are largely unknown.Due to its efficient perfusion, lipophilic milieu, and its richness in potential target structures, the adrenal gland appears to be susceptible to the effects of lipophilic EDCs (14).In a cohort study, BPA serum levels seemed to correlate with the diagnosis of non-functional adrenal incidentalomas (15).Additionally, in vivo studies revealed BPA's effect on neurobehavioral endpoints, and an underlying disruption of the hypothalamic-pituitaryadrenal axis, resulting in anxiety-like behavior (16,17).
The adrenal steroidogenesis underlies a complex regulation by superior hormones, involves multiple cellular organelles, and is dependent on the activity of cytochrome P450 and hydroxysteroiddehydrogenases.The various, sensitive steps in steroidogenesis can most likely be influenced by EDCs (18,19), keeping in mind that even minimal changes in steroid levels in vivo contribute to disrupted endocrine homeostasis.However, dose-response relationships and toxicological targets of EDCs on adrenal steroidogenesis remain still unclear (20).
Therefore, the present study comprehensively determines the effects of bisphenol A, F and S on the entire adrenal steroidogenesis, including gene expression of key enzymes in the NCI-H295R adrenocortical cell line.
Under microscopic control, cells were harvested every week using PBS (Sigma-Aldrich, St. Louis, Missouri, USA) and EDTAtrypsin (Sigma-Aldrich, St. Louis, Missouri, USA).Cell viability was assessed using the Countess II FL (life technologies, Carlsbad, California, USA).If not needed for further experiments, cells were distributed in relation 1:3 into new cell flasks.

Cell treatment
NCI-H295R cells were seeded in 96-well black plates (Corning Incorporated, Corning, New York, USA) with optically clear flat bottoms at a seeding density of 5000/well in 100μl total volume of complete medium.After 24 h of incubation at 37°C and 5% CO2, cells were exposed for 72 hours to the vehicle (1% DMSO), BPA, BPF, BPS, or the bisphenol mixture (each at 0.001, 0.05, 0.1, 0.25, 0.5, 1, 10, 25, 50, 100, 250, 1000μM).On the day of the experiment, DMSO-dissolved bisphenols were diluted in complete medium to the final concentration assuring the DMSO concentration was 1%.A total volume of 100μL was added to each well.
At the end of the incubation period, 150μL supernatant of eight parallel treated wells were collected, centrifuged at 800 rpm (23°C), and stored at -20°C until steroid quantification.

Cell viability assay
Cell viability was measured using the CellTiter-Glo ® Luminescent assay (Promega, Madison, Wisconsin, USA) following the manufacturer's protocol.After treatment, the cells were incubated with CellTiter-Glo ® Reagent for 10 min.After incubation, the luminescence of each well was determined using 1420 multilabel counter Victor 3 (Perkin Elmer, Waltham, Massachusetts, USA).
Each experiment was performed in three independent experiments in 8-plicates, and the results are expressed as fold changes to vehicle-treated cells.

Steroid hormone analysis
Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed using a Sciex 6500+ QTRAP (SCIEX, Framingham, USA) MS-system linked with an Agilent 1290 HPLC-system (G4226A autosampler, infinityBinPump, G1316C column-oven, G1330B thermostat; Santa Clara, USA).The quantification of 15 steroid hormones (aldosterone, androstenedione, corticosterone, cortisol, c o r t i s o n e , d e h y d r o e p i a n d r o s t e n d i o n ( D H E A ) , dehydroepiandrostendion-sulfate (DHEAS), 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, Dihydrotestosterone (DHT), estradiol, 17-hydroxyprogesterone (17-OHP), progesterone, and testosterone) via MRM was performed with the MassChrom-Steroids in Serum/Plasma ® IVDR conform kit (Chromsystems ® , Graüfelfing) as described in detail elsewhere (21).After off-line solid phase extraction of 500 mL cell supernatant, 15 mL were used for analysis.Concentrations were calculated with Analyst Software (1.6.3)via sixpoint calibration and 1/× weighting.Correctness of measurements was controlled by commercial quality controls and periodic participation in ring trails.

RNA Extraction and reverse transcription
For RNA extraction, NCI-H295R cells were seeded in 12 well plates (Corning Incorporated, Corning, New York, USA) at a density of 1x10^6 cells/well and exposed to 1% dimethyl sulfoxide (DMSO), BPA, BPF BPS, or the bisphenol mixture (each at 0.1, 1, 10, 25, 50μM).After 72 hours, cells were harvested using PBS and EDTA-trypsin.Cell pellets were centrifuged and washed with PBS.RNA was extracted using the Maxwell ® RSC simply RNA Tissue kit (Promega, Madison, Wisconsin, USA) following manufacturer's protocol, and stored at -80°C for later reverse transcription.
A total of 1μg RNA per sample was reverse-transcribed using the QuantiTect ® Reverse Transcription kit (Qiagen, Venlo, Netherlands) in a final volume of 40μL.cDNA was diluted by 1:10 in nuclease-free water and stored at -20°C for further analysis.

Quantitative real-time PCR
The RT-PCR reactions were performed using C1000 Thermal Cycler (biorad, Hercules, California, USA) with a reaction mixture comprised of 12.5μg water, 8μg TaqMan Gene Expression Master Mix (Thermo Fischer Scientific, Waltham, Massachusetts, USA), 8μg for each primer (Thermo Fischer Scientific, Waltham, Massachusetts, USA, see Supplementary Table 1), 2μg for each cDNA/well.Beta-Actin (ACTB) was used as a housekeeping gene.
mRNA levels were measured with quantitative real-time RT-PCR by the comparative Ct ( 2-DD Ct) method using qRAT expression analysis (22).
Each experiment was repeated three times in duplicates, and the results are demonstrated as fold changes in comparison to vehicletreated cells.

Statistical analysis
Statistical analysis was performed using GraphPad Prism Software (version 9.01 for Windows, GraphPad Software, Inc.).Data from cell viability, steroid quantification in response to bisphenol concentrations, and gene expression were analyzed using one-way ANOVA with Dunnett's post-hoc comparisons.Differences between vehicle-and BPA-/BPF-/BPS-or mixture-treated cells were analyzed using one-way-ANOVA and Dunnett*s post-hoc multiple comparisons.A p ≤ 0.05 was considered statistically significant.
Figures 1-3 were designed in GraphPad Prism.The graphical abstract and Figure 4 were created with biorender.com.

Cell viability
There was no significant difference in cell viability between untreated and vehicle-treated cells.Cell viability remained unaffected at lower bisphenol concentrations but decreased to 68% (p ≤ 0.001) by BPA, to 72% (p ≤ 0.001) by BPS and to 73% (p ≤ 0.001) by BPmix.For each substance, as well as for the mixture viability decreased to<66% (p ≤ 0.0001) at 250μM and to<6% (p ≤ 0.0001) compared to vehicle-treated control at 1mM.Calculated median lethal doses (LC 50 ) were 146.6μM for BPA, 263.6μM for BPF, 278.0μM for BPS, and 272.8μM for BPmix (Figure 1).

Steroid secretion
Concentrations, which caused pronounced reduction in cell viability, led to significant decrease in all steroid values as well.Therefore, the results obtained at these concentrations (250 μM and 1mM) are excluded from further analyses.Fold changes in steroid levels in the presence of 1nM to 100μM BPA, BPF, BPS, or bisphenol mixture (BPmix) are presented in Table 1 and Figure 2.

Steroid ratio
To identify potentially affected metabolic-enzymes in steroidogenesis, the ratios between the product and the substrate Illustration of substantial alterations on adrenal steroidogenesis, induced by an equimolar mixture of BPA, BPF, and BPS at doses >10µM.Enhanced conversions are displayed as bolder arrows, whereas inhibited steps are marked with a red X.Red and blue arrows indicate increased or decreased concentrations of the respective steroid.Some essential steroidogenic steps could not have been elucidated and are therefore marked with "?".Peripheral steroids, such as cortisone and DHT, are not shown.Created with biorender.com.
Aldosterone synthase activity, catalyzed by CYP11B2 seemed not to be affected by BPA, BPF, and BPS.However, the ratio of corticosterone to aldosterone was slightly reduced by BPmix (data not shown).
Regarding sulfotransferase activity of SULT2A1 no trends were detected for the individual treatments with BPA, BPF, BPS.However, the ratio of DHEA to DHEAS was slightly increased by BPmix (0.69 ± 0.01, p ≤ 0.0001 at 50μM; not shown).
5a-reductase, expressed by the ratio of testosterone to dihydrotestosterone, was not affected by BPA, BPF, BPS or BPmix.

Steroidogenic mRNA expression
mRNA expression of essential enzymes for adrenal steroidogenesis (StAR, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP21A2, HSD3B2) were examined in the NCI-H295R cells after 72 hours exposure to vehicle or bisphenols.Fold-changes of the mRNA of these genes are presented in Table 2.The most relevant changes were seen for StAR, CYP11B1, and CYP17A1.

Discussion
In the present study, comprehensive steroidomics, including a set of 15 steroids in bisphenol-exposed adrenocortical cells, were analyzed for the first time, paying special attention to a combination of BPA and its substitutes BPF and BPS.LC-MS/MS measurements revealed dose-dependent and substance-specific alterations of steroid secretion at different non-cytotoxic concentrations.Of note, many steroids decreased following bisphenol treatment, but the fact that some steroids were significantly increased clearly indicates a specific effect of bisphenols on the adrenal gland beyond any non-specific cytotoxic cell suppression.Substrate-to-product ratio calculations revealed strongly altered activity of key steroidogenic enzymes, probably based on multiple molecular mechanisms.
Reflecting "real life" conditions, where different contaminants can be detected in compounds, the combination of bisphenols A, F and S interfered with steroidogenesis resulting in elevated levels of 17-OH-progesterone, 21-deoxycortisol and estradiol.This combinatory treatment led to a steroidomic pattern as seen following BPF or BPS treatment only (see Figure 4), underlining that BPF and BPS, which are intended to replace BPA, may actually have a very relevant impact on adrenal steroidogenesis as well.
Additionally, all treatments led to significantly lowered androgens at non-cytotoxic doses, revealing that the known antiandrogenic activity of bisphenols extends to adrenal androgens, such as androstenedione and DHEA(S).
By calculating ratios of substrate and product of steroid metabolizing enzymes, dose-dependent alterations indicating an effect on the activity of these enzymes could be found.An inhibition of CYP17A1 associated 17,20-lyase activity and CYP21A2 can be certainly assumed, since substrate concentration outweighed the product concentration compared to control.Since these two crucial enzymes are dramatically inhibited, explaining the limited synthesis of effector hormones (e.g.cortisol), the accumulation of substrates (e.g.17-hydroxyprogesterone), and a potential "backlog" effect favoring alternative pathways like the formation of 21-deoxycortisol.
The data further suggest an enhanced activity of CYP11B1-and CYP19-mediated steps, as product concentrations increased in relation to substrate concentrations following bisphenol treatment.Regarding aromatase activity, BPA was found to induce CYP19A1 gene and protein expression levels, and further increased conversion activity, promoting estradiol synthesis in testicular Leydig cells (11).
Meanwhile, HSD3B2-related ratios were only affected by the individual treatment with BPA and the combinatory BPmix.Interestingly, the ratios reveal alterations in conversion dynamics at lower doses than expected when considering results of isolated substances, with trends starting at 500nM already.
As S ̌tefunkováet al. reported significantly enhanced levels of intracellular cholesterol after treatment with BPA, BPF, and BPS, this might be explained by either increased cholesterol uptake or synthesis, or an accumulation due to inhibited mitochondrial uptake via StAR and CYP11A1, which serves as the first step in steroidogenesis (12).This might explain, why most of the measured steroids are decreased within higher doses of treatment.However, elevated doses of certain hormones, such as 17-hydroxyprogesterone or 21-deoxycortisol, and elevated expression of the StAR enzyme seem to contradict an overall shutdown of steroidogenesis by bisphenols.
In accordance with previous studies (12, 18) describing significantly increased concentrations of progesterone following BPF exposure, treatment with BPF resulted in 7.3-, 11.2-, and 12.7fold increase at 30μM, 50μM, and 70μM BPF respectively.BPA led to lowered progesterone levels at >30μM (12, 18).However, in the current study, the effects (5.2-fold increase by 100μM BPF) were not as large as described before and, contrary to previous results, BPS did not alter progesterone significantly.Additionally, dramatically increased levels of 17-hydroxyprogesterone following BPF, BPS and BPmix exposure were seen.This might be of translational relevance, as administration of progestogenic contaminants during fetal development has led to virilization or feminization in animal models (23).
BPA and BPS exerted relevant anti-androgenic activity by significantly decreasing testosterone levels at doses >10μM.Additionally, BPF increased estradiol concentration at 100μM.Previous experiments detected similar results at low dose treatment with bisphenols, regarding lowered testosterone and increased estradiol (12,24).The data support the hypothesis of anti-androgenic activity of bisphenols, as not only testosterone, but also essential adrenal androgens (DHEA, DHEAS, and androstenedione) were affected by single and combined exposure.As adrenal androgens act as precursor hormones for the formation of testosterone and are key players in the adrenarche (21), the presence of antiandrogenic endocrine disruptors could influence puberty timing and sexual maturation (22).Estradiol increased following treatment with all tested bisphenols, while calculation of CYP19A1 kinetics clearly suggests an increased conversion of testosterone to estradiol.
While endocrine disruptive effects on the reproductive system and sex steroid homeostasis have been frequently reported, the mechanistic impact on key adrenal functionthe synthesis of gluco-and mineralocorticoidsis not conclusively understood.In our study, the most relevant glucocorticoid, cortisol was lowered by all tested bisphenols, while the mineralocorticoid aldosterone was affected only by BPA and BPS.This is in accordance with previous findings (18).While a decrease of most measured glucocorticoids (11-deoxycortisol, cortisol, and cortisone) could be detected, 21deoxycortisol, which plays a minor physiological role, was strongly elevated by treatment with BPF, BPS and BPmix.This might be explained by an inhibited activity of 21-hydroxylase (CYP21A2), which preferably converts 17-hydroxyprogesterone to 11deoxycortisol (25).However, its inhibition may promote the accumulation of substrates and alternative pathways.Therefore, 11b-hydroxylase activity (CYP11B1) and its expression could be enhanced by some bisphenols, leading to accumulation of 21deoxycortisol.However, an anti-glucocorticoid activity was seen at relevant doses >10μM.Interestingly in the present experiments, treatment with the BPmix led to additive changes compared to the respective mono-treatments, as the individual concentration in the mix was only a third of the concentrations in the separate approaches.Although expected due to the similar structure of the tested bisphenols, this strongly suggests an additive disruptive effect of bisphenols and further supports the obvious assumption that the EDC problem must not be solved by substitution with structurally similar substances.
In contrast to previous studies Klicken oder tippen Sie hier, um Text einzugeben., significant changes in gene expression of essential steroidogenic enzymes, such as rate-limiting CYP11B2, CYP19A1, CYP21A2, or HSD3B2 were only inconclusively observed.As mRNA levels of StAR, CYP11B1, and CYP17A1 were enhanced compared to control, by BPF and BPS, effects on gene expression seem to contribute to the disruption of adrenal steroidogenesis.
Since gene expression analysis does not correlate with steroid alterations, other mechanism should be considered.Also, nongenetic modes of action, such as (post-)translational effects, allosteric modulation, interaction with redox partners or phosphorylation status of steroidogenic enzymes, or oxidative stress of the mitochondria and smooth endoplasmatic reticulum involved in steroidogenesis, could be relevant.
For instance, a CYP17A1-inhibiting activity of BPA has already been suggested, affecting 17, 20-lyase and 17a-hydroxylase on a functional level.Zhang et al. showed the inhibition of 17,20-lyase activity, while 17a-hydroxylase seemed not to be affected in H295R cells in the presence of BPA (26,27).The present study supports this finding, as 17,20-lyase, converting 17-OH-progesterone to androstenedione, was significantly inhibited by BPF, BPS and BPmix, as respective ratios increased (see Figures 3D-F).Consistently, 17a-hydroxylase activity, representing the hydroxylation of progesterone, was either not relevantly altered by BPA, BPF (respective steroid ratios of around 1.0), or even enhanced by BPS and BPmix (ca.2-fold induction).Meanwhile, enhanced CYP17A1 expression might be a compensatory cellular mechanism to ensure steroid production, This dual effect suggests a mechanism that is not limited to gene/protein expression, as both functions of CYP17A1 would be affected in the same manner.As only the 17,20-lyase is dependent on P450 oxidoreductase and adequately phosphorylated serine and threonine residues (28), it is considerable that bisphenols interact with cytochrome b5 or the phosphorylation status of CYP17A1, thereby modulating its steroidogenic activity.As the activity of CYP21A2 is also highly dependent on its redox partner cytochrome b5 (29), and similar steroidomic trends are seen as with CYP17A1, this is consistent with bisphenols interfering with the steroidogenic pathways at enzymatic levels.
An accumulation of 17-hydroxyprogesterone and 21deoxycortisol can be seen in congenital adrenal hyperplasia, caused by a hereditary dysfunction of 21-hydroxlase (CYP21A2) (30).As steroidomic patterns after treatment with BPF, BPS and BPmix revealed the same accumulations of precursors (e.g.progesterone) or alternative steroids (e.g.21-deoxycortisol) and consequently lowered levels of mineralo-and glucocorticoid effector hormones, a specific blockade on CYP21A2-mediated steps seems to be obvious.
Furthermore, in silico analysis revealed the binding potential of BPA and its analogues to steroidogenic enzymes, which was comparable to CYP-inhibiting drugs, e.g.abiraterone.Most remarkable interactions were seen with CYP17A1, CYP21A2, and HSD3B2 (26).Thereby, allosteric modulation of steroidogenic enzymes by bisphenols is conceivable.
To draw a full picture, (31,32) Bisphenols are known to promote the generation of radical oxygen species, and consecutive mitochondrial dysfunction, as well as lipid peroxidation in steroidogenic tissues (33,34).Reduced mitochondrial activity was reported in the presence of bisphenols in H295R cells (12).If ROSmediated mitochondrial dysfunction might explain the altered activity of mitochondrial CYP-enzymes, such as CYP11B1, remains speculative and should be part of future research.
As a further explanation, disrupted signaling pathways and (in-) direct effect on steroidogenesis might explain the presented results.Lan et al. showed BPA-induced phosphorylation of the JNK/c-Jun pathway affecting corticosterone secretion in vivo (19).Besides, the involvement of bisphenols in the disruption of ERK signaling (35) or the MAPK pathway (36) has been reported.
In total, this study, in accordance with previous results, indicates that bisphenols do not affect adrenal steroidogenesis via a single mechanism, but more likely mediate their disruptive effects in multiple molecular ways, including gene expression and the modulation of enzymatic activity,

Limitations
It may be argued that concentrations of bisphenols above levels found in plasma or urine were used in this approach.On the other hand, it must be assumed that these substances accumulate in lipophilic tissue (such as the adrenal gland), especially following chronic exposureconsidering that bisphenols are detectable in >92% of the general European population (9) therefore, higher levels in adrenal tissue than measurable in the blood is plausible.Nonetheless, in vitro studies are unable to reflect the hormonal feedback mechanisms including hypothalamus, pituitary gland, and autocrine processes.Furthermore, metabolites of bisphenols, formed in vivo, were not included in the analysis.In total, cell culture studies methodologically lack an easy transferability to real life conditions in terms of duration and levels of exposure, but nonetheless they are a valuable tool to detect detailed endocrinedisrupting effects in human cellular pathways.

Conclusion
In conclusion, the extensive use and distribution of bisphenols over the last decades steers the attention to their potential influence on humans and the ecological systemespecially as global resilience levels on the burden of synthetic chemicals are trespassed (37).This study presents in a comprehensive steroidomic approach strong in-vitro evidence that bisphenols alter adrenal steroidogenesis in multiple molecular ways and several target structures in the steroidogenic pathway could be identified (e.g.CYP11B1, CYP17A1, CYP21A2).BPF and BPS were shown to significantly disrupt adrenal steroidogenesis, Therefore, their use as substitute compounds for BPA seems rather unreasonable.In the future, further studies will address the impact of the shown impacts on living organism to evaluate the long-term consequences for wildlife and human health.

TABLE 1
Fold changes of measured steroid concentrations after 72h of treatment with BPA, BPF, BPS, or BPmix.

TABLE 2
mRNA expression of genes of interest: Fold change of treatment compared to vehicle-treated control ± SD.