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Molecular and Cellular Endocrinology
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Molecular and Cellular Endocrinology
Evaluating the effects on steroidogenesis of estragole and trans-anethole in a feto-placental co-culture model
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Debbie Yancu*, Cathy Vaillancourt, J. Thomas Sanderson **
INRS - Centre Armand-Frappier Santé Biotechnologie, Laval, QC, H7V 1B7, Canada
ARTICLE INFO
Keywords:
Feto-placental unit Estragole Steroidogenesis Trans-anethole Aromatase Co-culture
ABSTRACT
In this study, we determined whether estragole and its isomer trans-anethole interfered with feto-placental steroidogenesis in a human co-culture model composed of fetal-like adrenocortical (H295R) and placental tro- phoblast-like (BeWo) cells. Estragole and trans-anethole are considered the biologically active compounds within basil and fennel seed essential oils, respectively. After a 24 h exposure of the co-culture to 2.5, 5.2 and 25 uM estragole or trans-anethole, hormone concentrations of estradiol, estrone, dehydroepiandrosterone, an- drostenedione, progesterone and estriol were significantly increased. Using RT-qPCR, estragole and trans-an- ethole were shown to significantly alter the expression of several key steroidogenic enzymes, such as those involved in cholesterol transport and steroid hormone biosynthesis, including StAR, CYP11A1, HSD3B1/2, SULT2A1, and HSD17B1, -4, and -5. Furthermore, we provided mechanistic insight into the ability of estragole and trans-anethole to stimulate promoter-specific expression of CYP19 through activation of the PKA pathway in H295R cells and the PKC pathway in BeWo cells, in both cases associated with increased cAMP levels. Moreover, we show new evidence suggesting a role for progesterone in regulating steroid hormone biosynthesis through regulation of the StAR gene.
1. Introduction
Essential oils are aromatic liquids isolated from the natural secre- tions synthesized by different plant organs and stored within specia- lized secretory cells (El Asbahani et al., 2015).
They are commonly extracted via steam or hydro-distillation from the plant materials. The extraction yields are extremely low, vary de- pendent on climate, soil composition, plant organ, age, and vegetative life cycle, and only 10% of plant species (termed aromatic plants) are able to produce essential oils (Svoboda and Greenaway, 2003).
One of the most popular forms of complementary therapies used by pregnant women is aromatherapy which employs essential oils for medicinal purposes (Hall et al., 2011; Joulaeerad et al., 2018). In light of this and recent evidence that has shown that certain essential oils can cross the fetal-placental barrier (Pelkonen et al., 2017), we thought it was important to shed some light on the effects of essential oils and potential risks they could pose to mother and fetus during pregnancy. A major concern regarding the safety of essential oils is that they are composed of hundreds of different compounds among which some may be working independently and others synergistically to mediate various in vitro or in vivo biological effects. Recently it has been reported that
basil and fennel seed are among the most popular medicinal herbs used during pregnancy (Nega et al., 2019; Trabace et al., 2015). Moreover, we have shown an effect of the essential oils of basil and fennel seed on fetal-placental steroidogenesis that we wanted to explore further (Yancu and Sanderson, 2019).
Essentials oils are composed of a complex mixture of volatile odorous compounds with good bioavailability, characterized by low molecular weight, and of which the majority are considered hydro- carbon terpenes and terpenoids. Despite the many constituents present within essential oils, sometimes up to 100 at various concentrations, they are typically categorized by their major components found at the highest concentrations (20-70%) according to analysis using gas chromatography and mass spectrometry (GC/MS). It is generally be- lieved that the most abundant compounds within the essential oil are responsible for its biological activity (Bakkali et al., 2008; Bayala et al., 2014).
In this study, we chose estragole and trans-anethole, the most abundant compounds within basil and fennel seed essential oils, re- spectively, to confirm our hypothesis that the steroidogenic effects ex- hibited by the oils are mediated by their predominant constituents. Using GC/MS data reports supplied by our essential oil manufacturer,
*Corresponding author. INRS-Centre Armand-Frappier Santé Biotechnologie, Laval, QC, H7V 1B7, Canada.
** Corresponding author. E-mail addresses: debbie.yancu@iaf.inrs.ca (D. Yancu), thomas.sanderson@iaf.inrs.ca (J.T. Sanderson).
we were able to quantify the amount of estragole and trans-anethole present within basil and fennel seed oils (74.61% and 76.53%, re- spectively) and individually assess those concentrations in a fetal-pla- cental co-culture model. Our main objective was to characterize the potential disruption of feto-placental steroidogenesis by estragole and trans-anethole. We focused on steroid hormone production, promoter- specific expression of CYP19, and aromatase catalytic activity with the goal of further delineating the signaling pathways involved in the steroidogenic response of the feto-placental co-culture to essential oils.
2. Materials and methods
2.1. Treatments
Active compounds of basil and fennel seed essential oils: estragole and trans-anethole, respectively, were obtained from Sigma-Aldrich (St- Louis, MO). Cells were exposed for 24 h to concentrations of 2.5, 5.2 and 25 uM estragole or trans-anethole (representing the concentrations found in 0.0005%, 0.001% and 0.005% basil and fennel seed oils, re- spectively, from Rocky Mountain Essential Oils (Orem, UT)) with or without the presence of 10 uM H89 (Sigma), selective PKA inhibitor, or 10 µM chelerythrine chloride (Sigma), selective PKC inhibitor, and 1 µM mifepristone, progesterone receptor antagonist (Thermo Fisher Scientific, Waltham, MA, USA). All compounds were dissolved in DMSO and final DMSO concentrations in co-culture medium never exceeded 0.1% for single treatments and 0.2% for co-treatments.
2.2. Cells of the feto-placental co-culture model
H295R human adrenocortical carcinoma cells (American Type Culture Collection (ATCC), Manassas, VA, no. CRL-2128) were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F12 (DMEM/ F12) without phenol red containing 1.2 g/L sodium bicarbonate and 2 mg/L pyridoxine HCl (Gibco, Luzern, Switzerland). Medium was completed with 2.5% NuSerum (VWR International, Radnor, Pennsylvania), 1% ITS (insulin, human transferrin, and selenous acid) + Premix (Thermo Fisher Scientific), and 1% penicillin/strepto- mycin (Gibco) as previously described (Thibeault et al., 2014). BeWo human placental choriocarcinoma cells (ATCC no. CCL-98) were cul- tured in DMEM/F12 without phenol red containing 1.2 g/L sodium bicarbonate and 2 mg/L pyridoxine HCl. Medium was completed with 10% fetal bovine serum (FBS; Hyclone, Tempe, AZ), and 1% penicillin/ streptomycin (Gibco). Co-culture set-up was as previously described (Thibeault et al., 2014), except for the replacement of 24-well culture plates with 6-well plates. For hormone quantification and RNA ex- traction under co-culture conditions, 7.5 x 105 H295R cells were seeded in 2 ml per well in 6-well plates and 3.5 x 105 BeWo cells were seeded in 1.5 ml in transwell inserts (polycarbonate membrane with 0.4 uM pores, Corning Life Sciences, Corning, NY). Importantly, both batches of cells were exposed to treated co-culture medium. All Ex- periments were performed using cells of 8-25 passage.
2.3. Cell viability
The toxicity of estragole and trans-anethole to H295R and BeWo cells was determined using WST-1 cell viability reagent (Roche, Basel, Switzerland) which is based on the cleavage of a tetrazolium salt by the mitochondria in metabolically active cells. Each cell type was seeded separately in 96-well plates at a density of 104 cells/well with 200 ul of appropriate culture medium. After 24h, cells were exposed to fresh medium containing increasing concentrations of estragole or trans-an- ethole for another 24 h. Cells were then incubated for 1.5 h with 20 ul of WST-1 reagent and the formation of formazan was measured using a SpectraMax M5 spectrophotometer (Molecular Devices, Sunnyvale, CA) at a wavelength of 450 nm.
2.4. Steroid hormone quantification
After a 24h exposure to estragole or trans-anethole, co-culture medium was collected. Hormone concentrations were determined by ELISA using assay kits obtained from DRG Diagnostics (Marburg, Germany) according to manufacturer’s recommendations; kits included estradiol (DRG Diagnostics EIA-2693), estrone (DRG Diagnostics EIA- 4174), progesterone (DRG Diagnostics EIA-1561), androstenedione (DRG Diagnostics EIA-3265), dehydroepiandrosterone (DHEA) (DRG Diagnostics EIA-3415) and estriol (EIA-3717).
2.5. RT-qPCR
Real-time quantitative PCR (RT-qPCR) was used to assess gene ex- pression of steroidogenic enzymes. After 24h of cell acclimatization following plating, cells were exposed for 24 h to the treatments. DMSO (0.1%) was used as a vehicle control. BeWo cells in the inserts were isolated separately from the H295R cells in the wells below. RNeasy mini kits (Qiagen, Mississauga, ON, Canada) were used to isolate RNA, which was then stored at - 80 ℃. RNA purity and quantification was determined with the 260/280 nm absorbance ratio (~2.0) using a Nanodrop (Thermo Fisher Scientific, Waltham, MA). RNA integrity was also assessed with Experion RNA chips (BioRad, Mississauga, ON, Canada). Reverse transcription was performed using 0.5 µg RNA with an iScript cDNA synthesis kit and T3000 Thermocycler (Biometra, Göttingen, Germany); resultant cDNA was stored at - 20 ℃. RT-qPCR was performed using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) with a CFX96 real-time PCR Detection System (Bio-Rad). Suitable reference genes for normalizing target gene expression were selected using geNorm software (Biogazelle, Zwijnaarde, Belgium) and all reference genes were combined to a geometric mean. All validated primer pair sequences are listed in Table 1.
2.6. Aromatase catalytic activity
Cellular CYP19 activity was assessed using a tritiated water-release assay as previously described (Lephart and Simpson, 1991; Sanderson et al., 2000). The amount of tritiated water released, as a measure of aromatase activity, was determined using a Microbeta Trilux liquid scintillation counter (PerkinElmer, Waltham, MA). Negative controls contained radioactive substrate but no cells. A positive control radio- active substrate was directly added to scintillation cocktail. Aromatase activity was expressed as a percent of control activity (DMSO). For- mestane (4-hydroxyandrostenedione) (1 µM), a selective aromatase in- hibitor, was used to verify the specificity of the assay for the enzymatic aromatization reaction.
2.7. Kinase activities and cAMP
Activation of PKA, PKC, and cAMP levels were evaluated using a PKA activity assay kit, PKC activity assay kit, and cAMP direct im- munoassay kit (Abcam, Cambridge, United Kingdom) following the manufacturer’s instructions. The co-culture was exposed to estragole or trans-anethole, with or without PKA inhibitor H89 (Sigma) or PKC in- hibitor chelerythrine chloride (Sigma), for 24 h. Cells were then lysed, instructions provided by assay kits, in the culture plates and the su- pernatants were collected after 15 min of centrifugation at 15,000 g. The samples were stored at - 80 ℃ prior to analysis of kinase activities or cAMP levels.
2.8. Statistical analysis
All data were analyzed using GraphPad Prism (version 5.04; GraphPad Software, San Diego, CA). Results are presented as means with standard errors (SEM) of three different experiments; per experi- ment each treatment was tested in triplicate. Statistically significant
| Steroidogenic Enzyme | Primer Pairs (5' -> 3') | Reference: |
|---|---|---|
| CYP19 coding region | Fw: TGTCTCTTTGTTCTTCATGCTATTTCTC Rv: TCACCAATAACAGTCTGGATTTCC | Sanderson et al. (2000) |
| CYP19 I.1 | Fw: GGATCTTCCAGACGTCGCGA Rv: CATGGCTTCAGGCACGATGC | Klempan et al. (2011) |
| CYP19 PII | Fw: TCTGTCCCTTTGATTTCCACAG Rv: GCACGATGCTGGTGATGTTATA | Heneweer et al. (2004) |
| CYP19 I.3 | Fw: GGGCTTCCTTGTTTTGACTTGTAA RV: AGAGGGGGCAAT TTAGAGTCTGTT | Wang et al. (2008) |
| CYP11A1 | Fw: CTTCTTCGACCCGGAAAATTT Rv: CCGGAAGTAGGTGATGTTCTTGT | Oskarsson et al. (2006) |
| CYP17 | Fw: AGCCGCACACCAACTATCAG Rv: TCACCGATGCTGGAGTCAAC | Kim et al. (2016) |
| HSD17B7 | Fw: CTGGAATGGCTCCGGGCTTTG C Rv: CCTGCCCTCGGAGACGGCGTCG | Shehu et al. (2011) |
| HSD17B1 | Fw: GTCTTCCTCACCGCTTTGCGCGCC Rv: GCACTGCGCCCGGCCTCGTCCTC | Takagi et al. (2017) |
| HSD17B4 | Fw: TGCGGGATCACGGATGACTC Rv: GCCACCATTCTCCTCACAACTC | von Krogh et al. (2010) |
| HSD17B5 | Fw: GGGATCTCAACGAGACAAACG Rv: AAAGGACTGGGTCCTCCAAGA | Xu et al. (2017) |
| HSD17B3 | Fw: AACGCACCGGATGAAATCCAGAGC Rv: GCCTGGCTACCTGACCTTGGTGTT | Qin & Rosenfield (2000) |
| HSD17B2 | Fw: CTGAGGAATTGCGAAGAACC Rv: AAGAAGCTCCCCATCAGTTG | Casey et al. (1994) |
| SULT2A1 | Fw: CCTCCAGCGGTGGCTACA Rv: AATCGTCCGACATGATGATGAC | Oskarsson et al. (2006) |
| STAR | Fw: TTGCTTTATGGGCTCAAGAATG Rv: GGAGACCCTCTGAGATTCTGCTT | Oskarsson et al. (2006) |
| MLN64 | Fw: CCTGCCCCGGTACCTCAT Rv: GCGCTGTCGCAGGTGAA | Borthwick et al. (2010) |
| HSD3B1 | Fw: GGAGATCAGGGTCTTGGACA Rv: CAGGCTCTCTTCAGGAATGG | Hogg et al. (2014) |
| HSD3B2 | Fw: TGCCAGTCTTCATCTACACCAG Rv: TTCCAGAGGCTCTTCTTCGTG | Kim et al. (2016) |
| SULT2A1 | Fw: TCGTCATAAGGGATGAAGATGTAATAA Rv: TGCATCAGGCAGAGAATCTCA | Shiraki et al. (2011) |
| CYP3A7 | Fw: CTCTTTAAGAAAGCTGTGCCCC | Kondoh et al. (1999) |
| Rv: GGGTGGTGGAGATAGTCCTA | ||
| Reference gene: | Primer Pairs (5' -> 3') | Reference |
| UBC | Fw: ATTTGGGTCGCGGTTCTTG Rv: TGCCTTGACATTCTCGATGGT | Vandesompele et al. (2002) |
| TBP | Fw: TGCACAGGAGCCAAGAGTGAA Rv: CACATCACAGCTCCCCACCA | Tratwal et al. (2014) |
| RPLO | Fw: GGCGACCTGGAAGTCCAACT Rv: CCATCAGCACCACAGCCTTC | Good et al. (2016) |
| RPII | Fw: GCACCACGTCCAATGACAT Rv: GTGCGGCTGCTTCCATAA | Radonic et al. (2004) |
| PBGD | Fw: GGCAATGCGGCTGCAA Rv: GGGTACCCACGCGAATCAC | Dolstra et al. (1999) |
differences from control (*p < 0.05; ** p < 0.01; *** p < 0.001) were determined using one-way analysis of variance (ANOVA) followed by a Dunnett post hoc test. We note that individual gene expression levels determined by RT-qPCR are relative to their own basal (DMSO) level of expression, therefore expression levels cannot be compared among genes or among promoter-specific CYP19 transcripts.
3. Results
3.1. The effects of estragole and trans-anethole on cell viability of H295R cells and BeWo cells
A 24 h exposure to estragole or trans-anethole did not significantly affect H295R (Fig. 1A) or BeWo cell (Fig. 1B) viability at concentrations up to 25 uM.
3.2. The effects of estragole and trans-anethole on steroid hormone concentrations in co-culture
Estragole and trans-anethole increased progesterone levels in co- culture by a statistically significant margin (Fig. 2A) at concentrations of 2.5 µM (33.2 ± 4.4 ng/ml and 32.3 + 2.4 ng/ml, respectively) and 5.2 µM (20.0 ± 3.9 ng/ml and 17.8 ± 0.9 ng/ml, respectively) compared to vehicle control, DMSO control (5.8 ± 0.5 ng/ml). There was no statistically significant change in progesterone levels at a 25 uM concentration of estragole or trans-anethole.
Estragole and trans-anethole significantly increased DHEA levels in co-culture at 2.5 uM (2.9 ± 0.4 ng/ml and 5.4 ± 0.2 ng/ml, respec- tively), 5.2 uM (3.6 ± 0.3 ng/ml and 5.0 ± 0.4 ng/ml, respectively) and 25 uM (3.4 ± 0.5 ng/ml and 5.1 ± 0.6 ng/ml, respectively) compared to DMSO control (1.0 ± 0.1 ng/ml) (Fig. 2B).
Androstenedione levels also increased significantly at estragole and trans-anethole concentrations of 2.5 uM (7.8 ± 0.4 ng/ml and 7.7 ± 0.1 ng/ml, respectively), 5.2uM (5.8 ± 0.7ng/ml and
A
150-
Estragole
B
150-
Estragole
Trans-anethole
BeWo Cell Viability (% DMSO control)
Trans-anethole
H295R Cell Viability (% DMSO control)
100-
100
-
50
50-
0
0
DMSO
2.5PM
5.2PM
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2.5PM
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Estragole
Estragole
Trans-anethole
Trans-anethole
Estragole
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Trans-anethole
40-
6-
Progesterone (ng/ml)
Androstenedione (ng/ml)
30-
8.
**
*
DHEA (ng/ml)
4.
**
**
**
6
**
20-
**
**
4.
10
2.
2-
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Estragole
Estragole
Trans-anethole
Trans-anethole
Estragole
15-
Trans-anethole
600
200-
**
**
Estradiol (pg/ml)
150
Estriol (ng/ml)
10-
**
400-
Estrone (pg/ml)
*
100
5.
200
50-
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2.5PM
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6.6 ± 0.3 ng/ml, respectively) and 25 uM (4.4 ± 0.3ng/ml and 4.7 ± 0.8 ng ml, respectively) compared to DMSO control (1.2 ± 0.1 ng/ml) (Fig. 2C).
Estradiol levels in co-culture rose significantly after treatment with estragole and trans-anethole at concentrations of 2.5 uM (472.0 ± 30.2 pg/ml and 397.3 ± 32.2 pg/ml, respectively), 5.2 uM (482. 6 ± 30.5 pg/ml and 367.8 ± 16.2 pg/ml, respectively) and 25 µM (470.0 ± 11.4pg/ml, and 290.5 ± 17.5 pg/ml, respectively) compared to vehicle control (91.8 ± 2.0 pg/ml) (Fig. 2D).
Estragole at concentrations of 2.5, 5.2 and 25 uM significantly in- creased levels of estrone in co-culture (184.2 ± 2.5 pg/ml, 173.4 ± 6.9 pg/ml and 165.1 ± 4.5 pg/ml, respectively) compared to DMSO control (134.8 ± 5.8 pg/ml) (Fig. 2E). However, only con- centrations of 2.5 and 5.2 uM trans-anethole significantly increased estrone levels (171.8 ± 8.1 pg/ml and 171.0 ± 2.7 pg/ml, respec- tively) (Fig. 2E).
Finally, we also detected significant increases in estriol levels after treatment with 5.2 uM and 25 uM estragole (8.4 ± 0.8 ng/ml and
9.8 ± 0.4 ng/ml, respectively) compared to vehicle control (4.0 ± 0.5 ng/ml) (Fig. 2F). Each concentration of trans-anethole sig- nificantly increased estriol levels (7.1 ± 0.6 ng/ml at 2.5 uM, 9.8 ± 0.9 ng/ml at 5.2 uM and 8.6 ± 0.8ng/ml at 25 uM) (Fig. 2F) compared to DMSO vehicle control.
3.3. The effects of estragole and trans-anethole on the mRNA expression of steroidogenic enzymes in H295R cells in co-culture
In H295R cells (representing the fetal compartment of the feto- placental steroidogenic unit), a 2-fold increase in PII-derived CYP19 mRNA expression levels was observed at an estragole concentration of 2.5 and 5.2 uM, and at a concentration of 2.5 uM trans-anethole, com- pared to DMSO control (Fig. 3A). StAR gene expression rose 5- to 13- fold compared to DMSO at estragole concentrations between 2.5 and 25 uM, with the lower concentrations producing the greater induction producing an inverted concentration-response curve (Fig. 3A). Simi- larly, trans-anethole significantly increased StAR gene expression levels
A
B
☒ CYP19 Coding region
CYP19 P2
CYP19 PI.3
Normalized Expression in H295R Cells
15
10
☒ StAR
Normalized Expression in H295R Cells
☒
CYP11A1
I
8
CYP17
SULT2A1
10-
6
CYP3A7
4
5.
**
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Normalized Expression in H295R Cells
15-
☒ HSD17B1
☒ HSD17B4
15
☒ HSD17B5
10-
☒
HSD17B7
☒ HSD3B1
10
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HSD3B2
5
*
**
*
*
*
M
**
*
0
89
-
8:
0
a
II
S
2
1
DMSO
2.5µM
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DMSO
2.5 HM
5.2PM
25PM
Estragole
Trans-anethole
by 2- to 8-fold in the 2.5-25 UM range with the greatest induction at the lowest concentration (Fig. 3B). We also detected significant increases in the mRNA levels of SULT2A1, and CYP3A7, which followed a classic concentration-response relationship with greatest induction at the highest concentrations. (Fig. 3A and B).
HSD17B5 mRNA levels rose between 4- and 17-fold at estragole concentrations in the 2.5-25 uM concentration range, whereas trans- anethole significantly induced HSD17B5 mRNA levels by 6- and 10-fold at 2.5 and 5.2 uM, respectively (Fig. 3C). In both cases, the lower concentrations of the compounds produced the greater induction. Es- tragole significantly increased mRNA levels of HSD17B1 and HSD17B7 in H295R cells (2-fold at 2.5 uM; 1.5-fold at 25 uM), whereas both es- tragole and trans-anethole increased HSD3B1 and HSD3B2 expression (Fig. 3D).
3.4. The effects of estragole and trans-anethole on the expression of steroidogenic enzymes in BeWo cells in co-culture
In BeWo cells, a 1.8-fold induction of I.1 promoter-derived CYP19 mRNA expression was seen at estragole concentrations in the 2.5 and 25 uM range (Fig. 4A), and a similar level of induction by 2.5 and 5.2 uM trans-anethole (Fig. 4B). However, while estragole produced a consistent effect on the expression of I.1-derived CYP19 transcripts, trans-anethole showed a reoccurring inverse concentration-response relationship.
CYP11A1 mRNA levels rose by 2.8-fold at 2.5 uM, 2-fold at 5.2 uM and 2.4-fold at 25 uM estragole (Fig. 4A). Trans-anethole increased CYP11A1 expression by 3.5-fold at 2.5 uM and 3-fold at 5.2 uM
(Fig. 4B).
Estragole and trans-anethole at concentrations of 2.5 and 5.2 uM increased HSD17ß1 mRNA levels by about 2.8-3.4-fold (Fig. 4C and D). Estragole and trans-anethole at concentrations between 2.5 and 25 uM significantly decreased HSD17B5 mRNA levels by 50% of DMSO control levels (Fig. 4C and D). MLN64 expression levels were reduced to 40-50% of DMSO levels for all concentrations of estragole and trans- anethole evaluated (Fig. 4A and B). Estragole at 2.5 uM increased HSD17B7 levels by 2.4-fold and trans-anethole increased its levels by 4.2 and 4.9-fold, respectively at 2.5 and 5.2 uM (Fig. 4C and D). Es- tragole increased HSD3B1 expression levels by 1.6-fold at 2.5 uM and 1.5-fold at 5.2 uM, whereas trans-anethole increased its expression by 1.5-fold at 2.5 uM (Fig. 4C and D).
3.5. Catalytic activity of aromatase
In order to confirm whether the effects of estragole and trans-an- ethole on the promoter-specific expression of CYP19 was associated with comparable changes in catalytic activity of the enzyme, each compound was tested for its ability to induce CYP19 activity in H295R and BeWo cells in co-culture. Estragole, at concentrations of 2.5 and 5.2 uM, increased CYP19 activity significantly in H295R (1.4- and 1.2- fold, respectively) and BeWo cells (1.3- and 1.2-fold, respectively) (Fig. 5A). Trans-anethole significantly increased CYP19 activity in H295R cells at 2.5 uM (1.4-fold) and in BeWo cells at 2.5 and 5.2 µM (1.3-fold) (Fig. 5B).
M
CYP19 Coding region
CYP19 PII
CYP19 1.1
MLN64
A
B
CYP11A1
CYP17
6
Normalized Expression in BeWo Cells
Normalized Expression in BeWo Cells
4
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OS
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T
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*
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Normalized Expression in BeWo Cells
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T
HSD17B1
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HSD17B4
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*
**
*
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DMSO
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3.6. Kinase activities and cAMP
To investigate whether estragole and trans-anethole increased the promoter-specific expression of CYP19 in H295R and BeWo cells (PII- and I.1-derived) via the PKA and/or PKC kinase pathways, the co-cul- ture was exposed to selective kinase inhibitors for 4 h prior to a 24 h
exposure to estragole and trans-anethole.
In the feto-placental co-culture, 2.5 uM estragole or trans-anethole increased PKC kinase activity by about 50% relative to DMSO control in both H295R and BeWo cells (Fig. 6A and B). PKC activity was not in- creased by estragole or trans-anethole in either cell type when the co- culture was pre-treated with PKC inhibitor, chelerythrine chloride (CC)
A
CYP19 activity (% control)
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150
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DMSO 2.5uM 5.2µM 25AM
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H295R Co-culture
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150
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H295R cells (co-culture)
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PKC activity (% of control)
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DMSO
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Estragole (5.2UM)
Estragole (25uM)
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Trans-anethole (25uM)
C
H295R cells (co-culture)
D
Bewo cells (co-culture)
PKC activity (% of control)
200
PKC activity (% of control)
200
b
b
b
b
150
a
150
a
a
a
a
a
100
100
50
50
0
0
DMSO
Chelerythrine Chloride
Estragole (2.5UM)
CC + Estragole (2.5UM)
Trans-anethole (2.5uM)
CC + Trans-anethole (2.5uM)
DMSO
Chelerythrine Chloride
Estragole (2.5UM)
CC + Estragole (2.5UM)
Trans-anethole (2.5UM)
CC + Trans-anethole (2.5UM)
(Fig. 6C and D).
Estragole and trans-anethole at 2.5 and 5.2 uM increased PKA ac- tivity by about 50% relative to DMSO control in H295R cells in co- culture (Fig. 7A). Estragole and trans-anethole did not affect relative PKA activity in BeWo cells in co-culture (Fig. 7B). PKA activity was not increased by estragole or trans-anethole in H295R cells after the co- culture was pre-treated with PKA inhibitor, H89 (Fig. 7C). Pre-treat- ment with H89 did not affect PKA activity in BeWo cells which remain unchanged relative to DMSO control (Fig. 7D).
Intracellular cAMP levels were increased by a 100% in both H295R and BeWo cells after treatment with estragole and trans-anethole at concentrations of 2.5, 5.2, and 25 uM (Fig. 8A and B).
To determine whether the rise in PKA and PKC activity was linked to the increases in promoter-specific expression of CYP19 we detected
in response to a 2.5 uM concentration of estragole or trans-anethole, we looked at changes to gene expression in the presence of CC and H89. After pre-treatment with CC, there was no significant change to pII- derived CYP19 expression in H295R cells (Fig. 9A). However, there was a significant reduction in pII-derived CYP19 transcripts after pre- treatment with H89 (Fig. 9A). In BeWo cells, there was a significant reduction in I.1-derived CYP19 expression in response to estragole and trans-anethole after pre-treatment with CC, while no change in CYP19 I.1 expression was detected after an H89 pre-treatment (Fig. 9B).
3.7. The effect of mifepristone on StAR mRNA expression and progesterone secretion
StAR protein is responsible for the rate-limiting step in
A
B
H295R cells (co-culture)
Bewo cells (co-culture)
PKA activity (% of control)
200
PKA activity (% of control)
150
**
**
**
*
150
100
100
50
50
0
0
DMSO
Estragole (2.5uM)
Estragole (5.2uM)
Estragole (25uM)
Trans-anethole (2.5UM)
Trans-anethole (5.2uM)
Trans-anethole (25uM)
DMSO
Estragole (2.5UM)
Estragole (5.2uM)
Estragole (25uM)
Trans-anethole (2.5uM)
Trans-anethole (5.2uM)
Trans-anethole (25UM)
C
D
H295R cells (co-culture)
Bewo cells (co-culture)
PKA activity (% of control)
200
b
PKA activity (% of control)
150
b
150
a
a
100
a
100
50
50
0
0
DMSO
H89
Estragole (2.5UM)
H89 + Estragole (2.51M)
Trans-anethole (2.5UM)
H89 + Trans-anethole (2.5HM)
DMSO
H89
Estragole (2.5UM)
H89 + Estragole (2.5mM)
Trans-anethole (2.5uM)
H89 +Trans-anethole (2.5mM)
steroidogenesis and by controlling the movement of free-cholesterol from the outer to the inner mitochondrial membrane. Along with the action of CYP11A1 and 3ß-HSD, progesterone is produced. Here, we set out to determine whether progesterone is involved in a positive feed- back mechanism regulating StAR gene expression. To do so, we co- treated the feto-placental co-culture with either estragole or trans-an- ethole and 1 µM of mifepristone, a progesterone receptor inhibitor. Mifepristone or a co-treatment of mifepristone with estragole or trans- anethole did not affect progesterone levels in co-culture compared to vehicle control DMSO (Fig. 10A).
In H295R cells in co-culture, the greatest induction of StAR mRNA levels occurred at 2.5 uM estragole or trans-anethole (Fig. 3A-B, 10B). Co-treatment with mifepristone resulted in a significant reduction of StAR mRNA levels, relative to treatment with estragole or trans-
anethole alone (Fig. 10B).
In BeWo cells in co-culture, StAR mRNA levels was significantly reduced at all concentrations of estragole and trans-anethole studied (2.5, 5.2 and 25 uM) (data not shown). Given that placental cells lack StAR protein, our interest was in MLN64 which possesses a closely re- lated gene sequence to StAR and may mediate the effects of StAR pro- tein in placental cells. MLN64 mRNA levels were also significantly re- duced by estragole and trans-anethole treatments (Fig. 10C). Co- treatment of estragole or trans-anethole with mifepristone resulted in no statistically significant difference in MLN64 mRNA levels to DMSO control (Fig. 10C).
A
H295R cells (co-culture)
B
Bewo cells (co-culture)
CAMP levels (% of control”
250
CAMP levels (% of control
200
**
*
**
**
*
**
**
**
**
200-
*
**
200
**
150
150
100
100.
50
50.
0
0
DMSO
Estragole (2.5UM)
Estragole (5.2UM)
Estragole (25uM)
Trans-anethole (2.5uM)
Trans-anethole (5.2uM)
Trans-anethole (25uM)
DMSO
Estragole (2.5UM)
Estragole (5.2UM)
Estragole (25UM)
Trans-anethole (2.5UM)
Trans-anethole (5.2uM)
Trans-anethole (25uM)
A
H295R cells (co-culture)
B
Bewo cells (co-culture)
Relative expression of PII-derived CYP19 transcripts
**
Relative expression of I.1-derived CYP19 transcripts
*
2.5
2.5
2.0·
2.0-
1.5
1.5.
1.0-
1.0
0.5-
0.5.
0.0
0.0
DMSO
Estragole
Estragole + CC
Estragole + H89
DMSO
Trans-anethole
Trans-anethole + CC
Trans-anethole + H89
DMSO
Estragole
Estragole + CC
Estragole + H89
DMSO
Trans-anethole
Trans-anethole + CC
Trans-anethole + H89
A
B
C
H295R/BeWo co-culture
H295R cells co-culture
BeWo cells co-culture
40-
15-
1.5
Progesterone (ng/ml)
Relative StAR expression
Relative MLN64 expression
a
Estragole
a
Trans-anethole
b
30-
10-
b
1.0-
a
20-
5
a
0.5
b
b
10
a
a
0
0
0.0
DMSO
2.5 FM
2.51M + Mifepristone
Mi fepristone
DMSO
Mi fepristone
Estragole 2.5pM
Trans-anethole 2.5pM
Estragole 2.5mM + Mifepristone
T-A (2.5}M) + Mifepristone
DMSO
Mi fepristone
Estragole 2.5µM
Trans-anethole 2.5pM
Estragole 2.5mm + Mifepristone
T-A (2.5H&M) + Mifepristone
4. Discussion
In this study, we used GC/MS reports supplied by our essential oil supplier to identify the biological compounds in basil and fennel seed essential oil that were present in the largest amounts, to test if these individual chemicals were responsible for any of the effects of the two essential oils on feto-placental steroidogenesis as it occurs in vivo (Sanderson, 2009) using a physiologically relevant co-culture system that mimics the interaction between the placental trophoblast, fetal
Fig. 10. (A) Progesterone production (mean ± SEM; n = 3) by H295R and BeWo cells in co-culture after a 24 h ex- posure to estragole or trans-anethole, in the presence or absence of 1 uM of the proges- terone receptor antagonist mifepristone. *** p < 0.001, determined by Student t- test. (B) Relative expression levels (mean ± SEM; n = 3) of StAR in H295R and (C) MLN64 in BeWo cells exposed for 24 h in co-culture to 2.5 uM estragole or trans-anethole, in the presence or absence of mifepristone. (a) No statistically sig- nificant difference between treatment and DMSO vehicle control; (b) A statistically significant difference between treatment and control (Student t-test; p < 0.05).
adrenal zone, and fetal liver (Thibeault et al., 2014). In addition, we wished to gain mechanistic insight into the role of each compound.
4.1. Steroid hormone levels, aromatase and kinase activities
Estragole and trans-anethole significantly increased levels of pro- gesterone, DHEA, androstenedione, estradiol, estrone, and estriol. The observed effects were not always concentration-dependent, such as for the effects on progesterone, androstenedione, and estradiol where the
lower concentrations of estragole and trans-anethole resulted in the greatest increases in these steroid hormones. This non-monotonic re- sponse was also seen for mRNA levels of steroidogenic enzymes in our feto-placental co-culture system. This was particularly true for the significant stimulation of promoter-specific expression of PII and I.1- derived CYP19 transcripts in H295R and BeWo cells, respectively, by both estragole (Figs. 3A and 4A) and trans-anethole (Figs. 3B and 4B). These non-monotonic effects were also seen in H295R cells in response to the neonicotinoids thiacloprid and thiamethoxam (Caron-Beaudoin et al., 2016), albeit in monoculture. It is possible that estragole and trans-anethole are activating intracellular signaling pathways that sti- mulate specific promoters involved in CYP19 expression at the lower concentrations we studied, but may start acting upon additional path- ways that impair or counteract this initial stimulation at higher con- centrations. These non-monotonic effects on the promoter-specific sti- mulation of CYP19 transcript were confirmed by our observation that the catalytic activity of CYP19 increased significantly only at the lowest concentrations of estragole and trans-anethole; at higher concentrations this initial increase in aromatase activity was no longer evident (Fig. 5A-B). Reports in Vandenberg et al. (2012) have shown that en- docrine-disrupting compounds in low doses can influence the response of an organ/system to endogenous hormones or even work synergisti- cally with other chemicals and natural hormones to create an additive response. Moreover, it is quite difficult to ascertain the biological re- levance of aromatase induction since we cannot measure the effect in rodents; which lack the CYP19 promoters unique to humans. Overall, a 1.2-1.4 fold induction of aromatase may not seem highly significant, we do not know in situ how this affects estrogen production. Moreover, pesticides have been recently shown to possess similar effects on ar- omatase (Caron-Beaudoin et al., 2016; Thibeault et al., 2014).
To investigate this non-monotonic response and to better under- stand the mechanism behind the ability of estragole and trans-anethole to stimulate aromatase catalytic activity and promoter-specific expres- sion of the CYP19 gene, we looked at changes in protein kinase activ- ities induced by our compounds. PKC activity was significantly in- creased in both H295R and BeWo cells in co-culture by 2.5 uM estragole or trans-anethole. The PKC signaling pathway is known to stimulate promoter I.1-derived CYP19 mRNA production in the placenta and associated BeWo cells (Hudon Thibeault et al., 2017; Klempan et al., 2011; Tan et al., 2013). Inhibition of PKC activity with chelerythrine chloride resulted in no significant change in PKC activity relative to DMSO in both H295R and BeWo cells and prevented the rise in I.1- derived CYP19 transcripts in response to 2.5 uM of estragole and trans- anethole detected in BeWo cells. It is important to note that cheler- ythrine chloride did not significantly alter PKC levels in DMSO-only treated cells, possibly because basal PKC levels are already low. To further understand the role played by estragole and trans-anethole at increasing promoter-specific CYP19 expression, we also assessed PKA activity and cAMP levels. A 2.5 and 5.2 uM concentration of estragole or trans-anethole increased PKA activity in H295R cells by about 50%; and this effect was inhibited by pre-treatment with H89. Moreover, pre- treatment with H89 prevented the rise in pII-derived CYP19 mRNA levels in response to 2.5 uM of estragole and trans-anehtole. The in- creased PKA activity in H295R cells by estragole and trans-anethole was associated with an increase in intracellular levels of cAMP, a second messenger known to activate PKA signaling. A previous study has shown that estragole and trans-anethole both significantly increase intracellular cAMP levels by 2- to 3-fold in rat smooth muscle cells (Henrique Bezerra Cabral et al., 2014). Both the PKA and PKC pathways can be activated by cAMP which leads to the phosphorylation of the cAMP responsive element binding protein 1 (CREB1). CREB1 then translocates to the nucleus and binds to a cAMP responsive-element which leads to the expression of CYP19 through PII/I.3 (Huang et al., 2011; Mao et al., 2007; Parakh et al., 2006).
Estragole and trans-anethole increased cAMP levels in BeWo cells in our co-culture model. It has been suggested that cAMP levels can rise as
a result of the activity of certain PKC isoforms in placental trophoblasts (Karl and Divald, 1996). These PKC isoforms are considered PMA-re- sponsive (phorbol 12-myristate 13-acetate) (Karl and Divald, 1996). Other studies have also shown that PKC activation can alter cAMP le- vels (Heyworth et al., 1985; Houslay, 1991). However, it is currently unclear which mechanisms allow the PMA-dependent increase in cAMP production, although it has been suggested to be caused by a direct effect on adenylate cyclase activity (Karl and Divald, 1996; Yoshimasa et al., 1987). More recent studies have shown that phorbol esters (Silinsky and Searl, 2003) and epidermal growth factor (EGF) (Connor et al., 1997) activate PKC in placental trophoblasts, but no information is available yet as to which PKC isoforms are involved and whether they are different from PMA-responsive isoforms. In all, more research is needed to better understand how PMA may increase cAMP levels in trophoblasts and which PKC isoforms are involved. In our study, our results suggests that trans-anethole and estragole are able to mimic PMA’s ability to stimulate adenylate cyclase and increase cAMP levels to activate PKC. It is also important to note that while all doses of es- tragole and trans-anethole that we evaluated increased cAMP levels, we only detected significant rises in PKA activity in H295R cells at estra- gole and trans-anethole concentrations of 2.5 UM and 5.2 uM and in- creases in PKC activity in BeWo cells at 2.5 uM of estragole and trans- anethole. We believe that at the lower concentrations of our treatments, CAMP levels are activating the kinase pathways we studied, while at higher concentrations of estragole and trans-anethole, the rise in cAMP could be acting on additional pathways that we have not yet been in- vestigated by either impairing or counteracting the initial stimulation of promoter-specific expression of CYP19. More work is needed to further explore the role that increased cAMP levels may be playing in the feto- placental co-culture outside of aromatase activation.
4.2. Progesterone regulates StAR gene expression
In H295R cells, we observed significant upregulation of the cho- lesterol transport gene StAR, whereas in BeWo cells MLN64 gene ex- pression was significantly reduced. In the placenta, cholesterol entry and placental steroidogenesis is thought to be regulated by MLN64 which shares 60% sequence homology with StAR (Esparza-Perusquía et al., 2015; Uribe et al., 2003). Recent studies that have also identified progesterone as a possible regulator of StAR in steroidogenic mouse Leydig cells (Schwarzenbach et al., 2003) and it is known that pro- gesterone receptors are expressed in H295R cells (de Cremoux et al., 2008). In our study, co-treatment of estragole and trans-anethole with mifepristone prevented estragole- and trans-anethole-mediated stimu- lation of progesterone production by the co-culture as well as the in- crease in StAR mRNA levels in the H295R cells of the co-culture. Mi- fepristone also prevented the estragole- and trans-anethole-mediated decrease in expression of the StAR homolog MLN64 in BeWo cells. Taken together, our results provide new evidence that progesterone appears to play an important role in regulating StAR gene expression in H295R and MLN64 gene expression in BeWo cells.
4.3. Estragole and trans-anethole disrupt steroidogenic enzyme expression
In addition to expression changes of CYP19 and StAR/MLN64 that we detected in the feto-placental co-culture in response to estragole and trans-anethole, there were several other steroidogenic enzymes that were affected by the treatment. In H295R cells, we detected a dose- dependent increase in the expression of SULT2A1 and CYP3A7 after both estragole and trans-anethole treatment. The fetal endocrine system relies on extensive conjugation of steroid hormones with sulfate, while the hormones are unconjugated in the placenta through sulfatase ac- tivity (Bradshaw and Carr, 1986; Geyer et al., 2017). Additionally, 16a-OH-DHEAS supplied by the fetal liver contributes to over 90% of placental estriol synthesis (Geyer et al., 2017). The latter explains the dose-dependent increase of estriol detected in co-culture. Moreover,
given the greatest increases in progesterone, DHEA, androstenedione, estrone, and estradiol levels detected at the lowest concentrations of estragole and trans-anethole evaluated, it is possible that the com- pounds may be activating sulfatase pathways that stimulate the release of conjugates DHEA-S from the fetal adrenal zone into placenta; an effect that is inhibited at higher concentrations. In H295R cells, we also observed increases in HSD3ß1/2 gene expression. Regulation of human 3ß-HSD enzymes is quite complex, involving multiple factors and re- ceptors, even dietary compounds have been shown to influence its ex- pression in various organs (Rasmussen et al., 2013). Future work would be needed to better understand the effect of estragole and trans-anet- hole on expression of HSD3ß1/2.
In BeWo cells, CYP11A1 gene expression was significantly increased at all concentrations of estragole and trans-anethole we studied. CYP11A1 cleaves the side-chain of cholesterol to form pregnenolone; the precursor for all steroid enzymes (Miller and Auchus, 2011) and since we noted increased hormone production in our co-culture, it is reasonable to suggest that this effect is linked.
There are currently twelve human 17ß-HSD enzymes characterized to date, some of which exhibit oxidative or reductive capabilities, thereby playing activating or inactivating roles (Luu-The, 2001). For the purpose of our study, we chose to evaluate changes in 17-HSD enzyme expression relevant to our feto-placental co-culture and to the individual cell lines, namely 17ß-HSD1, 17ß-HSD2, 17ß-HSD3, 17ß- HSD4, 17ß-HSD5, and 17ß-HSD7. Estragole and trans-anethole exerted the greatest effect on the expression of HSD17ß1 and HSD17ß7 in BeWo cells which are involved in the conversion of estrone into estradiol. Finally, estragole and trans-anethole significantly increased expression of HSD17ß5 in H295R cells and significantly decreased its expression in BeWo cells. 17ß-HSD5 is considered an androgenic enzyme since it enhances the production of testosterone from androstenedione. How- ever, we did not detect testosterone in co-culture and therefore do not consider this effect to be biologically relevant.
5. Conclusion
In this study, isomers estragole and trans-anethole, the most abun- dant chemical constituents of basil and fennel seed oil, respectively, disrupted feto-placental steroidogenesis in a co-culture model com- posed of fetal-like adrenocortical (H295R) and trophoblast-like (BeWo) cells. After a 24h exposure of the co-culture to estragole or trans-an- ethole (2.5, 5.2, and 25 uM), levels of estradiol, estrone, DHEA, an- drostenedione, progesterone and estriol were significantly increased; an effect that was not always concentration-dependent. Moreover, estra- gole and trans-anethole altered the gene expression of several key steroidogenic enzymes, including CYP11A1, HSD3B1, HSD3B2, HSD17B1, HSD17B5, HSD17B7, as well as the steroidogenic factor StAR which is involved in cholesterol transport. In the co-culture, estragole and trans-anethole increased PII- and I.1-mediated CYP19 expression in H295R and BeWo cells, respectively, as well as catalytic activity of the enzyme. Using selective protein kinase inhibitors, we were able to as- sociate increases in promoter-specific expression of CYP19 in H295R and BeWo cells to stimulation of PKA and PKC kinase activity, respec- tively. This study presented new evidence that progesterone appears to play an important role in regulating StAR gene expression in H295R and MLN64 expression in BeWo cells. Our results indicate that further study is necessary to determine the potential risks of using essential oils during pregnancy considering their potential to disrupt steroidogenic enzyme activity and expression in vitro.
Conflicts of interest
The authors declare to have no conflict of interest.
Acknowledgements
This work was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to JTS (grant no. 313313). We thank Élyse Caron-Beaudoin (Université de Montréal-School of Public Health) for her technical assistance with the co-culture and quantitative PCR analyses.
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