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Environmental Toxicology and Pharmacology
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ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY
Cis-bifenthrin inhibits cortisol and aldosterone biosynthesis in human adrenocortical H295R cells via cAMP signaling cascade
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Ye Yang ª,”, Chunlei Wangb, Hong Shenª, Hongliang Fanª, Jing Liu, Nanxiang Wuª
a School of Public Health, Hangzhou Medical College, Hangzhou 310013, China
b Department of Public Health, Yu Hang No.2 People’s Hospital, Hangzhou 311100, China
” Institute of Environmental Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
ARTICLE INFO
Edited by Dr. Alan Jeffrey Hargreaves
Keywords: Cis-bifenthrin Pyrethroid insecticides H295R cells Steroidogenic genes Cyclic adenosine monophosphate
ABSTRACT
Cis-bifenthrin (cis-BF) is a common-used pyrethroid insecticide frequently detected in environmental and bio- logical matrices. Mounting evidence highlights the endocrine disrupting effects of cis-BF due to anti-estrogenic or anti-androgenic activity. However, little is known about the exposure effects of cis-BF on adrenal cortex function. In this study, effects of cis-BF on biosynthesis of adrenal steroids, as well as the potential mechanisms were investigated in human adrenocortical carcinoma (H295R) cells. Cis-BF decreased basal production levels of cortisol and aldosterone, as well as cAMP-induced production of cortisol. Both he basal and cAMP-stimulated transcriptional levels of several steroidogenic genes were significantly down-regulated by cis-BF. As an impor- tant rate-limiting enzyme in steroidogenesis, the protein level of StAR was prohibited by cis-BF on both basal and cAMP-induced conditions. Intracellular level of cAMP was significantly reduced by cis-BF. Overall, these data suggest that cis-BF may inhibit the biosynthesis of cortisol and aldosterone via disrupting cAMP signaling cascade.
1. Introduction
Synthetic pyrethroids are widely used for pest control in agriculture, forestry, animal husbandry and indoor environment (Sparks and Nauen, 2015; USEPA, 2014). Due to widespread use, pyrethroid insecticides have been frequently detected in water, sediment, crops, seafood, indoor air, breast milk and urine (Affum et al., 2018; Bouwman et al., 2006; Garí et al., 2018; Feo et al., 2012; H. Li et al., 2016; Li et al., 2019). Cis-bifenthrin (cis-BF) is a highly effective and broad-spectrum type I pyrethoid commonly used for agricultural and public health applica- tions in recent decades. During 2008-2013, the worldwide sales of cis-BF jumped from 270 million, and ranked 12th-14th of the global insecticides market (Li, 2017). In human breast milk, the total concentrations of synthetic pyrethroids were detected to range from 87 to 1200 ng/g lipid weight (lw) , and the concentration of cis-BF reached 1.1-36 ng/g lw (Bouwman et al., 2006).
Increased use, as well as prevalent exposure of wildlife and human, has prompted growing concern over the potential adverse health effects of pyrethroid insecticides (Liu et al., 2011a, 2005; Wang et al., 2020; Yang et al., 2014; Ye and Liu, 2019; Zhang et al., 2020). A previous study found evidence for reduced sperm level, quality and motility in
association with urinary levels of pyrethroids metabolites (Meeker et al., 2008). A recent study also reported environmental pyrethroids exposure might adversely affect semen quality of reproductive-age men in Shanghai, China (Hu et al., 2020). Moreover, accumulating experi- mental studies also revealed pyrethroids had adverse effects on repro- ductive and endocrine system, and their major endocrine effects are known to be nuclear receptor-mediated (Liu et al., 2011a, 2011b; Ye and Liu, 2019; Solati et al., 2010; Zhang et al., 2018, 2016). In 1997, pyre- throid insecticides was listed as potential endocrine-disrupting chem- icals (EDCs) by United States Environmental Protection Agency (U.S. EPA) (Crisp et al., 1998; USEPA, 1997). Cis-BF is a well-known endo- crine disrupting pyrethroid insecticide. A few in vitro studies have suggested the potential of cis-BF to bind to sex hormone receptors and exhibit anti-estrogenic or anti-androgenic activity (Brander et al., 2012; Zhang et al., 2008). Several in vivo studies have suggested the disruption of cis-BF on production of sex hormones. For instance, in fish species, exposure to cis-BF induced the production of 170-estradiol and up-regulated expression of estrogen-responsive genes (Brander et al., 2012; Crago and Schlenk, 2015). Comparatively, in rat ovarian gran- ulosa cells, cis-BF significantly suppressed luteinizing hormone (LH)-induced prostaglandin E2 (Liu et al., 2011a).
* Correspondence to: 182 Tianmushan Road, Hangzhou 310013, China. E-mail address: yangye19870604@163.com (Y. Yang).
The adrenal cortex has been recognized as a vulnerable target organ for EDCs apart from gonads in endocrine system (Harvey and Everett, 2003; Hinson and Raven, 2006). Two major steroid hormones in adrenal cortex, cortisol and aldosterone, play an important part in multiple physiological processes. Cortisol is a primary glucocorticoid involved in a wide range of physiological processes such as glucose homeostasis, inflammation, hypersensitivity, immunosuppression and disease resis- tance (Kershaw and Hall, 2016). Excessive cortisol secretion can cause Cushing syndrome with symptoms of round face, centripetal obesity, high blood pressure and muscle and bone weakness (Roux et al., 2017), however, when cortisol secretion is insufficient, primary adrenal insuf- ficiency or Addison disease happens, causing symptoms of dizziness, fatigue, low blood pressure, and weight loss (Ross et al., 2010). Aldo- sterone is a most important physiological mineralocorticoid regulating sodium and fluid retention and potassium excretion (Lymperopoulos et al., 2009). Disordered aldosterone biosynthesis has been shown as a potential pathophysiological basis for diseases such as hypertension, heart failure, cardiac arrhythmias, arrhythmia, and metabolic and kid- ney diseases (Bertocchio and Jaisser, 2011; Catena et al., 2014; Mihai- lidou, 2012). Therefore, it is imperative to understand the disrupting effects of multiple EDCs on the biosynthesis of these two primary ad- renal steroids.
As an endocrine disrupting compound, cis-BF has been shown to interfere with the receptors of both glucocorticoid and mineralocorti- coid, exhibit antagonistic effects and also down-regulate glucocorticoid/ mineralocorticoid-responsive genes (Zhang et al., 2018, 2016). How- ever, to our knowledge, little is known about the exposure effects of cis-BF on the biosynthesis of glucocorticoid and mineralocorticoid, and the potential mechanisms also remains unclear. In this study, human adrenocortical carcinoma cells (H295R) were used to determine the disruption of cis-BF in basal and cAMP-induced production of cortisol and aldosterone. As well, to shed light on the potential mechanisms, the expression of enzymes involved in cortisol and aldosterone biosynthesis (Fig. S1), including steroidogenic acute regulatory (StAR), cholesterol side-chain cleavage enzyme (P450scc), 36-hydroxycortisol dehydroge- nase (36-HSD), 17«-hydroxylase (CYP17), 21-hydroxylase (CYP21), steroid 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), as well as the intracellular cAMP content was evaluated.
2. Materials and methods
2.1. Chemicals
The analytical standard of cis-BF (≥ 98%), 8-Bromoadenosine 3’,5’- cyclic monophosphate (8-Br-cAMP, ≥ 97%) and dimethyl sulfoxide (DMSO, ≥ 99.7%) were all purchased from Sigma (St. Louis, MO, USA). Stock solutions of cis-BF were prepared in DMSO and stored at - 20 ℃. All chemicals used in the present study were of analytical grade.
2.2. Cell culture
The human adrenocortical carcinoma H295R cell line (ATCC CRL- 2128, ATCC, Manassas, VA, USA) were kindly provided by Dr. Meir- ong Zhao in Zhejiang University of Technology, China. H295R cells were cultured in Dulbecco’s modified Eagle’s medium plus Ham’s F-12 nutrient mixture DMEM/F12 medium (Hyclone, Logan, USA) supple- mented with 1% penicillin-streptomycin (Gibco, Grand Island, NY), 1% insulin-transferrin-selenium-G (ITS-G, Gibco) and 1% Ultroser G (Pall Corporation, Port Washington, NY, USA) at 37 ℃ in a humidified, 5% CO2 atmosphere. The medium was refreshed three times a week, and the subculture was performed using 0.25% trypsin-EDTA (Gibco). Cells between passages 5 and 10 after thawing were used for exposure experiments.
2.3. Experimental design
Cis-BF was dissolved in DMSO, and 8-Br-cAMP, an analog of intra- cellular cAMP, was dissolved in sterile ddH2O and used to mimic the effects of adrenocorticotrophic hormone (ACTH) on steroidogenesis. H295R cells were seeded in culture plates, incubated in serum-free medium for 24 h, and then treated with 0, 1, 10, 100 nM of cis-BF diluted in phenol red-free medium (Gibco) in absence or presence of 1 mM 8-Br-cAMP for 24 h. The final concentration of DMSO in all treat- ments was 0.1% (v/v), and negative control group received 0.1% DMSO. Positive cells were exposed to medium containing 1 mM of 8-Br-cAMP and 0.1% DMSO. After 24 h exposure, the culture medium was collected for hormone measurement, and the cells were collected for gene expression, western blot and cAMP quantification.
2.4. Cell viability assay
To exclude the disruption of cell death on gene and protein expres- sion, the viability of H295R cells was measured. Specifically, H295R cells were seeded into 96-well plate at a density 3 x 105 cells/ml with 200 ul cell suspension per well, and then exposed to 0, 1, 10, 100 nM of cis-BF for 24 h. Cell viability was determined using CellTiter 96® AQueous One Solution Cell Proliferation (Promega, Madison, Wisconsin) as previously described (Liu et al., 2012). Briefly, after 24 h of exposure, 20 ul of the reagent was added into each well for an additional 4 h in- cubation, and the absorbance at 490 nm was recorded on the Infinite M200 plate reader (Tecan USA, Durham, NC).
2.5. Hormone measurement
H295R cells (3 x 105 cells/well) were seeded in 24-well plate. After 24 h of exposure, culture medium in each treatment was collected, centrifuged at 3000 rpm for 20 min, and the supernatant was used for hormone measurement. Quantification of cortisol and aldosterone in the supernatant was conducted using commercial Radioimmunoassay (RIA) kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s protocol. Hormone levels were deter- mined by recording the absorbance at 450 nm by a microplate reader (Tecan).
2.6. Gene expression assay
Gene expression analysis was performed as described previously (Yang et al., 2014). H295R cells were seeded in 24-well plate at s density of 3 × 105 cells/well. After 24 h exposure, total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, USA). The concentration of RNA was measured, and the first-strand complementary DNA was synthe- sized using the ReverTra Ace® qPCR RT kit (Toyobo, Osaka, Japan). Quantitative real-time polymerase chain reaction was set up with SYBR® Green PCR master mix (Toyobo) and carried out on Quant- Studio® 3 Real-Time PCR System (ThermoFisher Scientific Inc., Bris- bane) to determine the expression of key genes involved in steroidogenesis. The Oligonucleotide primers of genes were designed using OMIGA 2.0 software (Oxford Molecular Ltd., Madison, WI, USA) and presented in Table S1. The relative expression levels of genes were calculated using the 2-AACT method and normalized to the housekeeping gene ß-actin.
2.7. Western blot analysis
Western blotting analysis was performed as described previously (J. Liu et al., 2010). Briefly, cells (1.2 x 106 cells/well) were seeded in 6-well plate with 2.5 ml medium per well and cultured for 24 h. After 24 h exposure, cells were lysed, and the intracellular protein levels were extracted. Protein concentrations were determined using Bradford Pro- tein Assay Kit (Beyotime Biotech, Nantong, China). Protein extracts (50
ug) were separated on 12% SDS-PAGE gel and electrophoretically transferred onto a nitrocellulose membrane (0.2 um, Pall, USA). The membranes were blocked with 5% nonfat milk and then incubated with anti-StAR primary antibody (Cell signaling technology, 1:500) at 4 ℃ overnight. After being washed three times for 10 min, the membranes were immunoblotted with a HRP-labeled goat anti-rabbit IgG secondary antibody (Cell signaling technology, 1:1000) for 2 h at room tempera- ture under gentle agitation, and the blots were visualized using Novex™M ECL Chemiluminescent Substrate Reagent kit (Invitrogen) and detected on a Molecular Imager® Gel Doc™M XR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The optical density of the protein bands was quantified using Image Lab 5.2 software (Bio-Rad Laboratories, Inc.). The StAR protein level was standardized by comparison to ß-actin.
2.8. Quantification of cAMP
H295R cells were seeded in 12-well plate at s density of 6 x 105 cells/ well, and the level of intracellular cAMP was analyzed using a com- mercial Enzyme Linked Immunosorbent Assay (ELISA) Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, after 24 h exposure, the culture medium was removed, and the cells were washed twice using 0.9% NaCl and then lysed for 20 min in 1 ml of 0.1 M HCl at room temperature. The cell lysates were collected, transferred to 1.5 ml plastic Eppendorf vials and centrifuged at 1000g for 10 min. The su- pernatant was diluted 5-fold using sample diluent in the kit, and the following steps were conducted according to the manufacturer’s in- structions. The absorbance was recorded at 450 nm on a microplate reader (Tecan), and the cAMP content in each sample was calculated using a cAMP standard curve.
2.9. Statistical analysis
All experiments were repeated at least three times, and experimental data were expressed as mean ± standard error of mean (SEM) of three independent experiments with triplicates. All statistical analysis was carried out by using SPSS 16.0 (SPSS, Chicago, IL). The statistical dif- ferences between groups were tested by using one-way analysis of variance (ANOVA). If ANOVA revealed significant effects of treatments, the means were compared by Tukey’s post-hoc test, with p < 0.05 being considered significant.
3. Results
3.1. Effect on viability of H295R cells
No significant change in H295R cell viability was induced by cis-BF after 24 h exposure at concentrations of 1, 10, 100 nM when compared with the control group (data not shown). Thus, these doses were applicable for estimating the disrupted effects of cis-BF on steroido- genesis without causing cytotoxicity.
3.2. Effect on basal cortisol and aldosterone production
As shown in Fig. 1, cis-BF treatments caused notable decrease in basal production of cortisol and aldosterone. Compared with the control group, cortisol biosynthesis was significantly decreased by 24% and 41% in 10 and 100 nM cis-BF treatment groups respectively. Similarly, 10 and 100 nM cis-BF exposure significantly inhibited the production of aldosterone by 20% and 48% of the control respectively.
3.3. Effect on cAMP-induced cortisol and aldosterone production
Under exogenous stress or physiological stimuli, the secretion of ACTH in pituitary is increased to stimulate the production of adreno- cortical hormones to maintain homeostasis (Russell and Lightman, 2019). To understand the disrupted effects of cis-BF on the
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steroidogenesis regulation of ACTH under stress, H295R cells were treated with cis-BF in presence of 8-Br-cAMP which was used to mimic the stimulated effect of ACTH on intracellular cAMP.
As shown in Fig. 2, compared with the vehicle control (0.1% DMSO), 1 mM 8-Br-cAMP significantly elevated the biosynthesis of cortisol and aldosterone by 4.3-fold and 1.8-fold respectively. However, cis-BF diminished the cAMP-induced cortisol production in a dose-dependent manner, and the cortisol level in the 100 nM cis-BF treatment group was significantly decreased by 25% compared with 1 mM 8-Br-cAMP alone treatment group. Comparatively, 10 and 100 nM cis-BF respec- tively caused 8.2% and 10% decrease in aldosterone level compared to 8-Br-cAMP alone group, while no significant difference was observed.
3.4. Effect on mRNA expression of steroidogenic genes involved in cortisol and aldosterone biosynthesis
Steroid hormones including cortisol and aldosterone are synthesized from cholesterol by a serial of steroidogenic enzymes. Generally, following transportation of cholesterol through mitochondrial
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membrane by StAR, cortisol and aldosterone was converted from cholesterol via sequential reactions catalyzed by P450scc, 36HSD, CYP17, CYP21, CYP11B1 and CYP11B2 (Fig. S1) (Payne and Hales, 2004).
As shown in Fig. 3, exposure to 10 and 100 nM cis-BF caused sig- nificant down-regulation in basal mRNA levels of P450scc, 3ßHSD, CYP17 and CYP11B1 by 19% and 29%, 16% and 21%, 26% and 28%, 47% and 64%, respectively. In contrast, the basal mRNA levels of CYP21 and CYP11B2 were down-regulated by 43% and 40% only in 100 nM cis- BF treatment group. Of note, StAR was the most sensitive steroidogenic gene, and the basal transcription was significantly down-regulated in a dose-dependent manner, with 14%, 23% and 43% decrease of the con- trol at 1, 10 and 100 nM cis-BF groups, respectively.
3.5. Effect on cAMP-induced mRNA expression of steroidogenic genes involved in cortisol and aldosterone biosynthesis
As shown in Fig. 4, 8-Br-cAMP significantly stimulated the tran- scription of all steroidogenic genes involved in cortisol and aldosterone biosynthesis, and the mRNA levels of StAR, P450scc, 36HSD, CYP17, CYP21, CYP11B1, CYP11B2 in 8-Br-cAMP group were respectively increased by 3.4-, 2.8-, 1.4-, 6.3-, 1.8-, 4.5- and 4.3-fold relative to the vehicle control group. Consistent with the result of inhibitory effect on 8-Br-cAMP-induced cortisol production by cis-BF (Fig. 2), exposure to cis-BF at 10 and 100 nM significantly reduced 8-Br-cAMP-stimulated expression of most genes except P450scc and CYP11B2. Concretely, compared with 8-Br-cAMP group, 100 nM cis-BF down-regulated expression of 36HSD, CYP21, CYP11B1 by 45%, 22% and 40%, respectively. Exposure to 10 and 100 nM cis-BF caused down-regulation of StAR and CYP17 by 21% and 39%, 32% and 25% relative to 8-Br- CAMP group. P450scc transcription was decreased respectively by 10.3% and 16% in 10 and 100 nM cis-BF treatments compared with 8- Br-cAMP alone treatment, but no statistical difference was observed. No significant change was observed in the transcription profile of CYP11B2 by the addition of cis-BF at all tested concentrations.
3.6. Effect on basal and cAMP-induced StAR protein abundance
The protein StAR facilitates the transfer of cholesterol from outer to inner mitochondrial membranes, which is the rate-limiting and acutely- regulated process in steroidogenesis (Miller, 2017). As well, StAR is most susceptible to be inhibited by cis-BF among all steroidogenic genes based on the gene expression data (Figs. 3 and 4). Thus, the protein abundance of StAR in H295R cells were further tested.
Compared with the vehicle control, exposure to 10 and 100 nM cis- BF significantly down-regulated StAR protein expression by 36% and
47%, respectively (Fig. 5A). Exposure to 8-Br-cAMP dramatically induced the StAR protein expression by 5.7-fold of the control (Fig. 5B). However, the up-regulation of StAR protein level by 8-Br-cAMP was significantly inhibited by 100 nM cis-BF, with 31% decrease relative to 8-Br-cAMP group (Fig. 5B).
3.7. Effect on cellular cAMP levels
Data presented above showed cis-BF suppressed several steroido- genic genes. As most steroidogenic enzymes are cAMP-dependent, and changes in cAMP level may cause altered expression of downstream steroidogenic enzymes (Sewer and Waterman, 2001). Thus, the intra- cellular level of cAMP was further evaluated.
As shown in Fig. 6, exposure to 10 and 100 nM cis-BF caused sig- nificant reduction in the level of cAMP, with respective 25% and 21% decrease of the vehicle control. No significant change of cAMP level in 1 nM cis-BF exposure group compared with the control.
4. Discussion
In this study, a significant inhibition of adrenocortical hormones was found in H295R cells exposed to cis-BF, as shown by significant decrease in basal production of cortisol and aldosterone, as well as in cAMP- induced production of cortisol. The mechanism of inhibition was found to involve down-regulation of mRNA levels of several cAMP- dependent steroidogenic genes and decrease in protein abundance of StAR under both basal and cAMP-induced conditions, as well as reduced cellular cAMP level. These data indicated cis-BF might inhibit biosyn- thesis of cortisol and aldosterone via cAMP-dependent signaling cascade.
Release of glucocorticoids and mineralocorticoids exert a variety of actions under both basal and stress conditions to maintain or reestablish homeostasis (Bonnecaze et al., 2019). In this study, the basal production of cortisol and aldosterone was inhibited by cis-BF. Inhibition of these adrenal hormones indicated cis-BF might have a risk of primary adrenal insufficiency or other kidney diseases more or less (Ross et al., 2010). Consistently, previous studies also showed decrease in steroid hormones induced by other pyrethroid insecticides besides cis-BF. For instance, in female Sprague Dawley rats in diestrus, exposure to fenvalerate caused declined level of serum progesterone (He et al., 2006). Likewise, in primary cultured rat preantral ovarian follicles, the production of pro- gesterone, testosterone and estradiol was all inhibited by fenvalerate exposure (Fei et al., 2010). Data in the present study as well as previous studies indicated pyrethroid insecticides may have inhibitory effects on biosynthesis of steroid hormones.
Intracellular cAMP in adrenal cortex cells is generally accumulated
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in response to external or internal stress-induced activation of hypothalamus-pituitary-adrenal (HPA) axis, promoting the production of cortisol, aldosterone and other related hormones involved in man- aging stress, electrolyte balance, and blood pressure (Yan et al., 2018; Giordano et al., 2006). As described in previous studies, 8-Br-cAMP was added to mimic the induced effect of ACTH on steroidogenesis under stress conditions (Feng et al., 2016; Li et al., 2013). High concentration (100 nM) of cis-BF repressed 8-Br-cAMP-induced production of cortisol in H295R cells, indicating cis-BF might have inhibitory effect on ACTH stimulation of glucocorticoids production. As is known, gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are also pituitary hormones regulating steroidogenesis in a mechanism like ACTH, and similar inhibitory effect was observed in gonadal steroidogenic cells by cis-BF and other pyrethroids. For instance, in rat ovarian granulosa cells, exposure cis-BF blocked LH-inducible prostaglandin E2 and progesterone accumulation in the cultured medium (Liu et al., 2011a, 2011b). As well, another pyrethroid, fenvalerate, decreased FSH-induced production of both 17ß-estradiol and progesterone in rat granulosa cells (Chen et al., 2005).
The inhibitory effect of cis-BF on 8-Br-cAMP-induced cortisol pro- duction indicated a potential role of cAMP signaling cascade regulating biosynthesis of steroids in this suppressive effect (Furuta et al., 2008). To understand the mechanism of cis-BF effect on cortisol and aldosterone production, we further examined the expression of several major cAMP-dependent steroidogenic enzymes. StAR and P450scc are two critical enzymes that participate in the rate-limiting steps catalyzing the formation of pregnenolone, the precursor of cortisol and aldosterone (Miller, 2017). Pregnenolone is catalyzed by CYP17 to produce 17a-OH pregnenolone. Then, pregnenolone and 17«-OH pregnenolone are con- verted to progesterone and 17«-OH progesterone by 3HSD (Payne and Hales, 2004). Eventually, 17«-OH progesterone is catalyzed by CYP21 and CYP11B1 to produce cortisol, while the biosynthesis of aldosterone is catalyzed sequentially by CYP21, CYP11B1 and CYP11B2 from pro- gesterone (Payne and Hales, 2004). CYP11B1 and CYP11B2 is respec- tively involved in the final step of cortisol and aldosterone production (Wang et al., 2015).
In this study, the transcription levels of StAR and P450scc were significantly decreased by cis-BF at 1 nM and 10 nM, respectively. The 8- Br-cAMP-induced transcription of StAR was also inhibited by 10 nM and 100 nM cis-BF. Coherent with our result, previous studies found cis-BF inhibited LH-induced gene expression of StAR and P450scc in rat ovarian granulosa cells (Liu et al., 2011a, 2011b). Likewise, exposure to fenvalerate caused downregulted expression of StAR and P450scc in primary cultured rat preantral ovarian follicles (Fei et al., 2010). Apart
from pyrethroid insecticides, many previous studies reported suppres- sion of StAR and P450scc by other toxicants such as bisphenol A, 1H,1H, 2H,2H-perfluoro-decan-1-ol and chlorophenols which were suspected to disrupt steroidogenesis (Feng et al., 2016; C.S. Liu et al., 2010; Ma et al., 2011). These data, together with our results, indicate these two rate-limiting enzymes for steroidogenesis may be more sensitive to po- tential environmental endocrine disruptors.
As mentioned above, the enzymes 36HSD, CYP17, CYP21 and CYP11B1 are involved in the production of cortisol. In this study, both the basal and 8-Br-cAMP-stimulated transcriptional levels of these pro- teins were suppressed by 10 nM and 100 nM cis-BF. This result was consistent with the data showing decrease in both basal and 8-Br-cAMP- induced biosynthesis of cortisol. A previous in vivo study has also found inhibited transcription of 36HSD in male rats by another pyrethroid insecticide, allethrin, leading to decreased steroids levels (Madhubabu and Yenugu, 2017). However, to our knowledge, this is the first study comprehensively investigating the disrupted effects of pyrethroid in- secticides on these steroidogenic enzymes. Among these steroidogenic genes, CYP17, a critical enzyme responsible for cortisol production, was comparatively more susceptible as cis-BF exerted inhibitory effect on CYP17 transcription even at the concentration of 10 nM. In accordance with our findings, the basal or 8-Br-cAMP-stimulated transcription of CYP17 was significantly down-regulated by other potential EDCs, such as bisphenol analogs, pentachlorophenol, nitrophenols and per- fluorinated chemicals, along with decreased levels of steroid hormones (Feng et al., 2016; C.S. Liu et al., 2010; Ma et al., 2011; Furuta et al., 2008). Given the important role of these enzymes in steroidogenesis, we speculate the inhibitory effect of cis-BF on cortisol and aldosterone in this study may partially due to down-regulated transcription of these steroidogenic enzymes.
The enzyme CYP11B2 is only involved in the biosynthesis of aldo- sterone and catalyzes the final step (Payne and Hales, 2004). The results showed that the basal expression of CYP11B2 was reduced by cis-BF at the highest concentration of 100 nM, while no significant inhibitory effect was found on 8-Br-cAMP-induced transcriptional level, similar to the transcription data of P450scc. As P450scc and CYP11B2 are both involved in the biosynthesis of aldosterone (Payne and Hales, 2004; Wang et al., 2015), we speculate the non-detected inhibitory effect of cis-BF on cAMP-induced aldosterone production might be partially due to the non-significant change in expression of P450scc and CYP11B2 compared with 8-Br-cAMP alone treatment group. Nevertheless, we consider higher concentration of the pyrethroid may cause significant change in transcription of these enzymes and production level of cAMP-induced aldosterone, which needs further investigation.
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Among all steroidogenic genes, StAR catalyzes the first rate-limiting step of steroids biosynthesis, mobilization of cholesterol from outer to inner mitochondrial membrane, which is acute and rapid (Bizzarri et al., 2017). Targeted gene disruption of StAR caused disorders in the biosynthesis of steroid hormones in mice, accompanied with adreno- cortical insufficiency and florid lipid deposits in adrenal cortex (Caron et al., 1997a, 1997b). In this study, StAR was shown to be most sensitive in comparison with other steroidogenic genes, evidenced by data that basal transcription was significantly reduced by cis-BF even at the con- centration of 1 nM. Thus, the protein level of StAR was further evalu- ated. A dose-dependent decrease was observed in both basal and
cAMP content (% of control)
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CAMP-induced protein level of StAR following cis-BF exposure. Down-regulation of StAR could inhibit the biosynthesis of steroid hor- mones via reducing the availability of cholesterol, the precursor sub- stance, for the first conversion reaction of steroidogenesis occurring in the inner mitochondrial membrane (X. Li et al., 2016). Thus, down-regulation of StAR at both transcriptional and translational levels by cis-BF might play an important part in inhibiting the production of cortisol and aldosterone.
CAMP is one of the most important intracellular second messengers which is responsible for the biosynthesis of steroid hormones including cortisol and aldosterone (Ruggiero and Lalli, 2016). The transcription of most steroidogenic genes such as StAR, P450scc, CYP17, 36HSD, CYP21 and CYP11B are modulated by cAMP (Sewer and Waterman, 2001). The cAMP/protein kinase A (PKA) signaling pathway plays a critical role in the biosynthesis of cortisol and aldosterone (Wang et al., 2015). Recently, increasing evidence has highlighted that cAMP-dependent signaling pathway is an important target for potential endocrine dis- ruptors. Several chemicals, such as perfluorooctyl iodide, pentachloro- phenol, 2,4,6-trichlorophenol, organotin and 8:2 FTOH, have been reported to interfere with steroidogenesis via cAMP signaling cascade (Yan et al., 2018; Wang et al., 2015; J. Liu et al., 2010; C.S. Liu et al., 2010; Ma et al., 2011). In the present study, cis-BF at 10 nM and 100 nM significantly decreased intracellular cAMP level by 25% and 21%, respectively. This result was in accordance with data of down-regulated transcription level of steroidogenic genes and decreased protein level of StAR. Therefore, we speculated the inhibited production of the intra- cellular cAMP might cause suppression of cAMP/PKA cascade, which might be the primary cause for inhibited biosynthesis of cortisol and aldosterone.
In conclusion, this study demonstrated that cis-BF could inhibit the biosynthesis of cortisol and aldosterone in H295R cells. The mechanisms of this inhibitory effects might involve suppression of cAMP/PKA signaling cascade, evidenced by decreased intracellular cAMP level and down-regulation of steroidogenic genes. Our findings may provide knowledge of endocrine disrupted effects of pyrethorid insecticides on adrenal gland and the potential mechanistic basis. Further in vivo studies are needed to confirm the findings, as well as to investigate the toxicological effects of pyrethroids and evaluate their potential health risks to wildlife and humans.
CRediT authorship contribution statement
Ye Yang: Conceptualization, Methodology, Validation, Writing -
original draft, Writing - review & editing, Funding acquisition, Project administration. Chunlei Wang: Resources, Investigation, Formal anal- ysis, Visualization, Writing - review & editing. Hong Shen: Data cura- tion, Supervision, Investigation, Writing - review & editing. Hongliang Fan: Supervision, Investigation, Writing - review & editing. Jing Liu: Validation, Writing - review & editing. Nanxiang Wu: Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by Zhejiang Provincial Natural Science Foundation of China (LY17B070009) and Zhejiang Provincial Science and Technology Program (2017F30003) and Zhejiang Medical and Health Science and Technology Project (2017KY036).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.etap.2021.103784.
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