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Toxicology Reports
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toxicology reports
Conazole fungicides inhibit Leydig cell testosterone secretion and androgen receptor activation in vitro
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Maarke J.E. Roelofs a,b,*, A. Roberto Temmingª, Aldert H. Piersma b,a, Martin van den Bergª, Majorie B.M. van Duursen a
a Endocrine Toxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht, The Netherlands
b Center for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands
ARTICLE INFO
Article history: Received 24 March 2014 Received in revised form 12 May 2014 Accepted 12 May 2014 Available online 22 May 2014
Chemical compounds studied in this article: Cyproconazole (PubChem CID: 86132) Fluconazole (PubChem CID: 3365) Flusilazole (PubChem CID: 73675) Hexaconazole (PubChem CID: 66461) Myclobutanil (PubChem CID: 6336) Penconazole (PubChem CID: 91693) Prochloraz (PubChem CID: 73665) Tebuconazole (PubChem CID: 86102) Triadimefon (PubChem CID: 39385) Triticonazole (PubChem CID: 6537961)
Keywords: Androgen receptor (AR) Conazole fungicides
ABSTRACT
Conazole fungicides are widely used in agriculture despite their suspected endocrine dis- rupting properties. In this study, the potential (anti-)androgenic effects of ten conazoles were assessed and mutually compared with existing data. Effects of cyproconazole (CYPRO), fluconazole (FLUC), flusilazole (FLUS), hexaconazole (HEXA), myconazole (MYC), pencona- zole (PEN), prochloraz (PRO), tebuconazole (TEBU), triadimefon (TRIA), and triticonazole (TRIT) were examined using murine Leydig (MA-10) cells and human T47D-ARE cells sta- bly transfected with an androgen responsive element and a firefly luciferase reporter gene. Six conazoles caused a decrease in basal testosterone (T) secretion by MA-10 cells varying from 61% up to 12% compared to vehicle-treated control. T secretion was concentration- dependently inhibited after exposure of MA-10 cells to several concentrations of FLUS (IC50 = 12.4 [M) or TEBU (IC50 = 2.4 [M) in combination with LH. The expression of steroido- genic and cholesterol biosynthesis genes was not changed by conazole exposure. Also, there were no changes in reactive oxygen species (ROS) formation that could explain the altered T secretion after exposure to conazoles. Nine conazoles decreased T-induced AR activation (IC50S ranging from 10.7 to 71.5 M) and effect potencies (REPs) were calculated relative to the known AR antagonist flutamide (FLUT). FLUC had no effect on AR activation by T. FLUS was the most potent (REP=3.61) and MYC the least potent (REP=0.03) AR antago- nist. All other conazoles had a comparable REP from 0.12 to 0.38. Our results show distinct in vitro anti-androgenic effects of several conazole fungicides arising from two mecha- nisms: inhibition of T secretion and AR antagonism, suggesting potential testicular toxic
Abbreviations: 30-HSD1, 3ß-hydroxysteroid dehydrogenase type 1; 17ß-HSD3, 17ß-hydroxysteroid dehydrogenase type 3; AR, androgen receptor; BMR, benchmark response; cAMP, 8-bromoadenosine 3’,5’-cyclic monophosphate; CHO cells, Chinese hamster ovary cells; Cyp11A1, cytochrome P450 enzyme 11A; Cyp17, cytochrome P450 enzyme 17; CYP19, cytochrome P450 enzyme 19 (aromatase); CYP51, cytochrome P450 enzyme 51/lanosterol 14x- demethylase; CYPRO, cyproconazole; DMEM, Dulbecco’s Modified Eagle Medium; EC50, half maximal effective concentration; EDCs, endocrine disrupting chemicals; FLUC, fluconazole; FLUS, flusilazole; FLUT, flutamide; FP, forward primer; FSH(R), follicle-stimulating hormone (receptor); H295R, human adrenocortical carcinoma cells; HEXA, hexaconazole; HMG-CoA red, HMG-CoA reductase; HSD(s), hydroxysteroid dehydrogenase(s); IC50, half maximal inhibitory concentration; LH(R), luteinizing hormone (receptor); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MYC, myclobu- tanil; NCBI, National Center for Biotechnology Information; PBS, phosphate-buffered saline; PEN, penconazole; Por, cytochrome P450 oxidoreductase; PRO, prochloraz; REP, relative effect potency; RIA, radioimmunoassay; ROS, reactive oxygen species; RP, reverse primer; RT-qPCR, real time quantitative polymerase chain reaction; StAR, steroidogenic acute regulatory protein; T, testosterone; TEBU, tebuconazole; TRIA, triadimefon; TRIT, triticonazole.
* Corresponding author at: Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht, The Netherlands. Fax: +31 30 2535077.
E-mail address: m.j.e.roelofs@uu.nl (M.J.E. Roelofs).
http://dx.doi.org/10.1016/j.toxrep.2014.05.006
effects. These effects warrant further mechanistic investigation and clearly show the need for accurate exposure data in order to perform proper (human) risk assessment of this class of compounds.
1. Introduction
Several studies indicate a global decline in human male fertility over the past decades due to poor semen quality, a suggested decline in sperm count, and low- ered testosterone levels in men [1-3]. Furthermore, an overall increase up to 12% in assisted reproductive treat- ments is observed in Scandinavian countries as well as Switzerland, The Netherlands, and United Kingdom over the past years [4]. In 20% of infertile couples this infertility was attributed to male factors solely and in another 30-40% male factors are conducive [5]. Exposure to environmental chemicals, including endocrine disrupting chemicals (EDCs), is often suggested to be an impor- tant contributing factor to these trends in male infertility [6,7].
Among the list of suggested EDCs pesticides are strongly represented [8]. Conazoles are a class of azole-based fungi- cides that are widely used as pesticides in the cultivation of crops [9] but also as human and veterinary pharma- ceuticals for the treatment of oropharyngeal, vaginal, as well as systemic candida and mycosis infections [10]. These compounds decrease fungal membrane integrity by inhibiting the cytochrome P450 enzyme lanosterol 14x- demethylase (CYP51), which is essential for ergosterol biosynthesis and maintaining proper membrane fluidity and permeability in fungi [9]. Besides fungal CYP51, cona- zoles also target CYP51 of mammals and other vertebrates, which catalyzes the formation of the cholesterol precursor zymosterol [11,12]. Conazoles are known to have in vivo endocrine disruptive effects in mammals. For instance, demasculinization of male rat fetuses occurred upon in utero exposure to several conazoles [13]. Yet, it remains to be investigated to what extent the known effects of a few tested conazoles are reminiscent for the whole group of conazoles.
The testicular microenvironment is pivotal for mam- malian steroidogenesis and intratesticular androgens are required for normal spermatogenesis [14]. In adult males, spermatogenesis is driven by the gonadotropins luteiniz- ing hormone (LH) and follicle-stimulating hormone (FSH). Via activation of the LH receptor (LHR), LH stimulates testosterone (T) production in the Leydig cells. Testicular production of T in interstitial Leydig cells is prerequisite for proper spermatogenesis and involves multiple steroido- genic enzymes, e.g. steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1), 17a-hydroxylase/20-lyase (CYP17A1), 3ß- and 17ß-hydroxysteroid dehydrogenase (3ß-HSD and 17ß-HSD, respectively) [15]. Subsequently, testosterone binds to the androgen receptor (AR) present in Sertoli cells, which, in combination with FSH binding to the FSH recep- tor (FSHR), stimulates the progression of spermatogenesis [16].
Conazoles are known to inhibit the steroidogenic enzyme aromatase (CYP19) in several tissues and cell lines, which is involved in the conversion of androgens to estrogens [9,10,17-19]. Conazoles also cause catalytic inhi- bition of the CYP17 enzyme, responsible for the conversion of pregnenolone and progesterone to androgen precur- sors, in the human adrenocortical carcinoma H295R cell line and porcine adrenal cortex microsomes [20]. Previ- ous work in H295R cells showed a decrease in T secretion after exposure to econazole, epoxiconazole, ketoconazole, miconazole, prochloraz, propiconazole, and tebuconazole [10]. In combination with the drop in T secretion, an increase in progesterone biosynthesis was seen after expo- sure to prochloraz, indicating that the role of the CYP17 enzyme is very important in this matter [21]. Further- more, Cyp26A1, a crucial enzyme within in the retinol metabolism pathway, seems to be a target for conazoles in the zebrafish embryo [12], an underlying mechanism for developmental toxicity. Spermatogenesis is tightly regu- lated by several steroidogenic processes involving multiple enzymatic conversions. The production of steroids by conversion of cholesterol via a cascade of several (CYP) enzymes is the first and crucial step to initiate sperm production, which makes it a vulnerable target for EDCs interference.
In spite of the large production and extensive usage of many conazoles, accurate data on human exposure lev- els are scarce. Besides occupational and pharmaceutical exposure, individuals can also be exposed to conazoles by environmental, food, resident, or bystander expo- sure. This is supported by increasing concentrations of conazole pesticides found in surface and waste waters [22]. According to case reports on the risk assessment of tebuconazole, conazoles are moderately and chron- ically toxic to aquatic species. The environmental fate route is mainly via the soil, where it is persistent due to its elimination half-life of approximately 800 days [23]. Pesticide usage surveys performed in the UK show that triazole usage has increased from 6.1 in 1990 to approx- imately 16.4 million ha treated in 2011 [24]. Among the conazoles, tebuconazole (2.5 million ha) is the most fre- quently used conazole fungicide, followed by prochloraz and cyproconazole (both 1.3 million ha), and then flusila- zole and triticonazole (0.6 and 0.5 million ha, respectively). Because of this extensive usage of conazoles, there is a potential risk that humans and wildlife are frequently, possibly chronically exposed to these compounds via their environment. The potential to affect steroid hor- mone synthesis in combination with the likelihood of frequent exposure make conazoles an important and rel- evant group of compounds to consider for effects on male fertility.
In this in vitro study, the effects of ten conazoles on two key male reproductive factors were assessed and
| Conazole | Abbreviation | Type | Use | Structure |
|---|---|---|---|---|
| Cyproconazole | CYPRO | Triazole | Pesticide | N N CI N |
| OH | ||||
| CH3 F | ||||
| Fluconazole | FLUC | Triazole | Pharmaceutical | N F N 1 N N N OH N N N N |
| Flusilazole | FLUS | Triazole | Pesticide | H3 C. Si F F |
| N CI N N | ||||
| Hexaconazole | HEXA | Triazole | Pesticide | CI OH |
| CH3 | ||||
| CN H3C | ||||
| Myclobutanil | MYC | Triazole | Pesticide | N N 1 N |
CI
Table 1 (Continued)
| Conazole | Abbreviation | Type | Use | Structure |
|---|---|---|---|---|
| N N N | ||||
| H3C | ||||
| Penconazole | PEN | Triazole | Pesticide | CI |
| CI N N CI | ||||
| Prochloraz | PRO | Imidazole | Pesticide | O O N |
| H3 C CI CI | ||||
| CI H3C CH3 | ||||
| Tebuconazole | TEBU | Triazole | Pesticide | H3C OH N |
| N N | ||||
| O O CH3 | ||||
| Triadimefon | TRIA | Triazole | Pesticide | CI CH3 CH3 N N |
| N N N | ||||
| Triticonazole | TRIT | Triazole | Pesticide | N OH CI H3C H3C |
compared, namely testicular steroidogenesis and AR response. These conazoles were selected based on their usage as a fungicide for crop protection and included cypro- conazole (CYPRO), flusilazole (FLUS), hexaconazole (HEXA), myclobutanil (MYC), penconazole (PEN), prochloraz (PRO), tebuconazole (TEBU), triadimefon (TRIA), and triticonazole (TRIT) (Table 1). For comparison, we also included one
conazole used as pharmaceutical, i.e. fluconazole (FLUC; Table 1). Effects on basal and LH-stimulated T secretion and steroidogenic gene expression were studied in murine MA-10 Leydig cells. A number of toxicants is also known to contribute to a decrease in sperm viability and motility by increasing ROS production in the testis and epididymis [25]. Therefore, effects of conazoles on ROS formation in
MA-10 cells were determined as well. In addition, effects on AR activation were assessed in human T47D-ARE cells stably transfected with a luciferase reporter gene.
2. Materials and methods
2.1. Chemicals
The ten selected conazoles (Table 1) were purchased from Sigma-Aldrich Co. (Zwijndrecht, The Netherlands): cyproconazole (CYPRO; 99.8%, CAS# 94361-06-5), flu- conazole (FLUC; ≥98%, CAS# 86386-73-4), flusilazole (FLUS; 99.8%, CAS# 85509-19-9), hexaconazole (HEXA; 99.7%, CAS# 79983-71-4), myclobutanil (MYC; 99.3%, CAS# 88671-89-0), penconazole (PEN; 99.1%, CAS# 66246-88-6), prochloraz (PRO; 99.1%, CAS# 67747-09-5), tebucona- zole (TEBU; 99.6%, CAS# 107534-96-3), triadimefon (TRIA; 99.7%, CAS# 43121-43-3), and triticonazole (TRIT; 98.8%, CAS# 131983-72-7). SU10603 was a kind gift from Dr. Honora Cooper Eckhardt (Hovartis Pharmaceuticals Cor- poration, Summit, USA). Stock solutions were prepared in DMSO resulting in a maximal solvent concentration of 0.1% (v/v) in the exposure medium.
2.2. MA-10 Leydig cell culture
The murine Leydig tumor cell line MA-10 was kindly provided by Dr. Mario Ascoli (University of Iowa, Iowa City, IA, USA) [26]. Cells were cultured as described previously by Dankers et al. [27]. In short, cells were grown in 1:1 Dulbecco’s Modified Eagle Medium/F-12 nutrient mixture (Ham) with phenol red (DMEM/F-12 1:1, #11320; Gibco, Life Technologies Europe BV, Bleiswijk, The Netherlands) supplemented with 15% HyClone (#SH30068.03; Thermo Fisher Scientific, Waltham, MA, USA), 2% HEPES [1 M] (#15630; Gibco), and 1% penicillin/streptomycin (#15140; Gibco) and maintained at 37 ℃ in a humidified atmosphere (95%) with 5% CO2. Cells were cultured twice weekly and culture medium was refreshed 24h prior to subculturing. Flasks and plates were coated at room temperature with 0.1% gelatin (Attachment Factor Protein; Gibco) 45 min prior to use.
2.3. Testosterone secretion assay
T secretion was assessed with MA-10 cells plated at a density of 2.0 x 105 cells/well in 24-well Plates 24h prior to exposure. 8-Bromoadenosine 3’,5’-cyclic monophos- phate (cAMP; [100 [M]) induces the expression of genes of steroidogenic enzymes and was used as positive con- trol. SU10603 [1 µM] is a catalytic CYP17 enzyme inhibitor [28] and was used as a control for decreased T secre- tion. For basal T measurements cells were exposed to the selected conazoles [10 [M] alone. Gonadotropin LH (10 ng/ml =8.5 IU/mL) was used to stimulate the Leydig cells to produce T. To determine the effect of selected cona- zoles on LH-induced T secretion, cells were exposed to a combination of LH (10 ng/mL) and SU10603 (0.05-1 µM), FLUS or TEBU (0.3-10 µM). After a 48-h exposure, medium was collected and stored at -20℃ until further use. T measurements in the media were performed with a commercially available T radioimmunoassay (T RIA)
kit according to the manufacturer’s instructions (#DSL- 4900; analytical sensitivity = 0.18 pg/mL; Beckman Coulter GmbH, Krefeld, Germany).
2.4. Gene expression
For gene expression experiments, MA-10 cells were plated at a density of 6.0 x 105 cells/well in 12-well Plates 24h prior to exposure. Cells were exposed for 6h to CYPRO, FLUS, PRO, TEBU [10 [M], and the positive con- trol cAMP [100 [M]. Total RNA was isolated from exposed MA-10 cells by chloroform-phenol extraction using RNA InstaPure according to the manufacturer’s instruction (Eurogentec, Liège, Belgium). Purity and concentration of isolated RNA was determined spectrophotometrically at absorbance wavelengths of 230, 260, and 280 nm using a NanoDrop2000 Spectrophotometer (Thermo Fisher Sci- entific, Waltham, MA, USA). RNA samples were diluted to a concentration of 66.7 ug/mL and stored at -80℃ until further use. cDNA was prepared using the iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Veenendaal, The Netherlands) and synthesized cDNA was diluted to the appropriate concentration for each primer pair (Supple- mentary Table 1). Real time quantitative polymerase chain reaction (RT-qPCR) was performed with a mixture contain- ing 7.5 pL iQ SYBR green supermix (Bio-Rad Laboratories Inc., Veenendaal, The Netherlands), 0.6 L forward primer (FP) and 0.6 pL reversed primer (RP) [each 10 [M], 0.3 µL RNAse free water, and 6 L of diluted cDNA.
Supplementary Table 1 related to this article can be found, in the online version, at doi: 10.1016/j.toxrep. 2014.05.006.
The expression of five steroidogenic genes was studied: steroidogenic acute regulatory protein (StAR), cytochrome P450 enzyme 11A1 (Cyp11A1), cytochrome P450 enzyme 17 (Cyp17A1), 3ß-hydroxysteroid dehydrogenase type 1 (3ß-HSD1), and 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3). Also, the expression of three cholesterol biosynthesis genes was studied: cytochrome P450 enzyme 51 (Cyp51), HMG-CoA reductase (HMG-CoA red), and cytochrome P450 oxidoreductase (Por). ß-Actin was used as a reference gene. Sequences of the primer pairs used are depicted in Supplementary Table 1. All primers span an exon-exon junction to ensure mRNA amplification only and were run through National Center for Biotechnol- ogy Information (NCBI) Blast (nucleotide non-redundant database) to confirm specificity. Efficiency was determined and was for all primer pairs between 90 and 110%. The mixtures were placed in the CFX Connect™M (Bio-Rad Labo- ratories Inc.) and firstly heated till 95 ℃ for 3 min, following 40 cycles of denaturation at 95℃ for 15 s and anneal- ing/extension at 60℃ for 45 s. Subsequently, a melt curve was run to ensure the exclusion of primer dimers and other non-specific products formed during the RT-qPCR. Gene expression of each sample was expressed as threshold cycle (Ct), normalized to the reference gene ß-actin (ACt), and fold induction relative to the DMSO control was calculated.
2.5. Reactive oxygen species assay
ROS production in MA-10 cells was assessed using the fluorescent dye H2-DCFDA (#D-399; Gibco). MA-10 cells
were plated at a density of 7.5 x 104 cells/well in 96-well Plates 24h prior to addition of the fluorescent dye. Cells were loaded with H2-DCFDA [10 [M] for 2 h prior to expo- sure at 37℃. After loading, the dye was removed and cells were washed twice with warm PBS. Subsequently, cells were exposed to the ten selected conazoles at con- centrations ranging from 0.01 to 100 µM for up to 48 h. Dye and exposure solutions were prepared in serum- free assay medium (DMEM/F-12 1:1 without phenol red, #11039; Gibco). Fluorescence was measured spectropho- tometrically at wavelengths of 485/530 nm (Infinite M200 microplate; Tecan Group Ltd., Männedorf, Germany) at T= 0 (to determine the basal background level), 1, 24, and 48 h of exposure. As a positive control for oxidative stress at the short time point (T= 1 h) H2O2 [20 mM] (hydrogen perox- ide 30%, #107209; Merck KGaA, Darmstadt, Germany) was used, for the longer time points (T=24 and 48 h) rotenone [100 [M] (#45656; Sigma-Aldrich Co.) was used. As non- exposed control cells show a basal ROS production over time, data are expressed as average percentage compared to the time-matched control values.
2.6. T47D-ARE cell culture
The human breast cancer cell line T47D-ARE was kindly provided by Prof. Dr. Michael Denison (University of California, Davis, CA, USA). T47D-ARE cells are trans- fected with an androgen responsive element with a firefly luciferase reporter gene [29]. Cells were grown in Dul- becco’s Modified Eagle Medium with phenol red containing 4.5 g/L D-glucose, L-glutamine, and pyruvate with phe- nol red (DMEM, #41966; Gibco) supplemented with 10% fetal bovine serum (FBS, #10270; Gibco) and 1% peni- cillin/streptomycin (#15140; Gibco) and maintained at 37 ℃ in a humidified atmosphere (95%) with 5% CO2. Cells were sub-cultured twice every week.
2.7. Androgen receptor reporter gene assay
Culture medium of T47D-ARE cell was replaced by assay medium 72 h prior to seeding. Assay medium was composed of Dulbecco’s Modified Eagle Medium without phenol red containing 4.5 g/L D-glucose (DMEM, #31053; Gibco) supplemented with 10% HyClone (#SH30068.03; Thermo Fisher Scientific), 1% L-glutamine [200 mM] (#25030; Gibco), 1% sodium pyruvate [100 mM] (#11360; Gibco), and 1% penicillin/streptomycin (#15140; Gibco). Cells were seeded at a density of 4.0 x 105 cells/well in white 96-well plates with a clear flat bottom (#655098; Greiner Bio-One, Alphen aan den Rijn, The Netherlands) 48 h prior to exposure. AR activation was determined by measuring the luciferase reaction luminescence. The lumi- nescent signal evoked by the luciferase reaction was mea- sured as relative luminescence units (RLU) of T47D-ARE cells exposed to concentration curves ranging from 100 pM to 100 MM of the ten selected conazoles. Exposures were performed in the presence or absence of EC50 of T [20 nM]. The known AR antagonist flutamide (FLUT) was used as positive control for AR antagonism [30]. After a 24-h expo- sure, cells were washed with warm phosphate-buffered
saline solution (PBS, diluted 1:10 with sterile water, #14200; Gibco) and incubated with 1x luciferase cell cul- ture lysis reagent (pH=7.8; E1531; Promega, Madison, WI, USA) for 30 min. Subsequently, luciferase activity was measured by addition of reagent mix (LUMIstar Galaxy luminometer, BMG Labtech GmbH, Ortenberg, Germany). The reagent mix was composed of tricine [20mM] (#T5816; Sigma-Aldrich Co.), (MgCO3)4Mg(OH)2-5H2O [1.07 mM] (#227668; Sigma-Aldrich Co.), MgSO4.7H2O [2.67 mM] (#63138; Sigma-Aldrich Co.), EDTA [0.1 mM] (#ED2SS; Sigma-Aldrich Co.), DTT [33.3mM] (#D9779; Sigma-Aldrich Co.), coenzyme A [261 [M] (#A2181; Sigma-Aldrich Co.), luciferin [470 [M] (#1605; Promega), and ATP [530 [M] (#10127531001; Roche Diagnostics Cor- poration, Indianapolis, IN, USA) dissolved in Milli-Q water (pH=7.8).
For FLUT and each conazole exposure the half max- imal inhibitory concentration (IC50) of AR activation was derived from concentration-response curves using a sigmoidal dose-response nonlinear regression curve fit with variable slope following the formula (1):
y = E0 + (Emax x X”1) bn + Xn
. ) (1)
In the above Hill equation, y is the dependent variable (AR response), X the independent variable (exposure con- centration), E0 the estimated background response level, Emax the maximum response, b the computed half maxi- mal inhibitory concentration of flutamide (IC50;FLUT), and n the shaping parameter of the Hill curve.
For each conazole the concentration, i.e. benchmark response (BMR), needed to elicit 25% of the inhibitory effect on AR activation response caused by flutamide (BMR25;FLUT) was calculated by using the formula (2) below:
BMR25%FLUT “conazole X”
= 10{-[(log((Emax/(y-Eo))-1))/n]+log(IC50”conazole X”)} (2)
Subsequently, relative effect potencies were calculated for each conazole relative to flutamide (REPFLUT) using the respective BMRs in the following formula (3):
REP “conazole X” = (
BMR25% FLUT BMR25% FLUT “conazole X” ) (3)
2.8. Cytotoxicity
Cell viability of MA-10 and T47D-ARE cells after exposures was determined by measuring the capac- ity of cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) to formazan by the mitochondrial enzyme succinate dehydrogenase. After exposure, remaining medium was removed and cells were incubated with MTT (1 mg/mL) for 30 min at 37℃ in a humidified atmosphere (95%) with 5% CO2. After aspira- tion, 1 mL isopropanol was added at room temperature in order to extract the formed blue colored formazan [31]. Absorbance was measured spectrophotometrically at a wavelength of 595 nm (POLARstar Galaxy, BMG Labtech GmbH).
A
3.0×104
*
[T] (% of DMSO control)
2.5×104
2.0×104
150
100
*
50
*
*
*
*
*
*
0
DMSO
LH
SU10603 CYPRO
FLUC
FLUS
HEXA
MYC
PEN
PRO
TEBU
TRIA
TRIT
B
150
+ SU10603 + LH
[T] (% of LH control)
O. FLUS + LH
*
TEBU + LH
100
2
*
50
*** 4
*
*
0
*
LH
-7
-6
-5
compound [log M]
2.9. Data analysis
All experiments were performed in triplo and within each independent experiment each concentration was tested in duplicate (T secretion assays), triplicate (gene expression experiments and AR reporter gene assays), or quadruplicate (ROS assays). The results are depicted as the mean of replicates of each experiment with standard error (SEM). Data calculations were performed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance of differences of the mean as com- pared to the control was calculated using a two-tailed unpaired Students’ t-test (for single concentrations) or a one-way ANOVA and post hoc Dunnett’s test (for concen- tration curves). Differences with P<0.05 were considered statistically significant.
3. Results
3.1. Testosterone secretion inhibition
In order to study the possible effects of conazoles on male sex steroid production in Leydig cells, basal T secretion by MA-10 cells was assessed after a 48-h exposure to the selected conazoles (10 p.M). This concen- tration did not significantly affect MA-10 cell viability (data not shown). Basal T secretion by vehicle-treated control
(0.1%, v/v DMSO) MA-10 cells was 0.24±0.09 pg/mL. Cells exposed to the positive control for increased T secretion (100 µM cAMP) showed a significant increase of 875-fold in secreted T levels in the medium compared to medium of vehicle-treated cells (data not shown). Exposure to the gonadotropin LH (10 ng/ml = 8.5 IU/mL) caused a sta- tistically significant increase of 215-fold in T secretion compared to vehicle-control cells (Fig. 1A). Exposure to the CYP17 inhibitor SU10603 (1 µM) statistically significantly decreased T secretion by 82% compared to vehicle-treated cells (Fig. 1A). Of the ten selected conazoles (10 µM), CYPRO (61%), FLUS (49%), HEXA (36%), PRO (23%), TEBU (12%), or TRIT (44%) statistically significantly inhibited T secretion by MA-10 cells compared with vehicle-control (Fig. 1A).
To evaluate the effect of conazoles on LH-induced T secretion, MA-10 cells were exposed to various concen- trations of two widely used conazoles that also showed marked basal T secretion inhibition, i.e. FLUS and TEBU, alone or in combination with LH (10 ng/ml). We selected both FLUS and TEBU as representatives of the group of conazoles tested because of the extensive literature on developmental and reproductive toxic effects available, their high usage, as well as their ability to decrease basal T secretion. SU10603, FLUS, and TEBU all concentration- dependently inhibited T secretion (Supplementary Fig. 1). In combination with LH, SU10603, FLUS, and TEBU inhibited T secretion with IC50 values of 0.5, 12.4, and
A
60
*
40
gene expression (fold of DMSO control)
CAMP
CYPRO
FLUS
PRO
TEBU
30
20
*
10
10
*
5
0
StAR
Cyp11A1
Cyp17
3-HSD1
17ß-HSD3
B
3
CAMP
CYPRO
FLUS
PRO
TEBU
gene expression (fold of DMSO control)
*
2
*
*
*
1
*
0
Cyp51
HMG-COA red
Por
2.4 M, respectively (Fig. 1B). To determine the extent of inhibition by FLUS and TEBU, the relative effect potency (REP) of these conazoles was calculated relative to the known CYP17 inhibitor SU10603 (REPSU10603 ) according to the formula (4):
IC50;SU10603
REPSU10603 “conazole X” = IC50 “conazole X” ) (4)
This calculation resulted in a REPSU10603 of 0.04 for FLUS and 0.21 for TEBU.
Supplementary Fig. 1 related to this article can be found, in the online version, at doi:10.1016/j.toxrep.2014.05.006.
3.2. Expression of steroidogenic genes
Next, we investigated whether inhibition of T secretion by four of the most extensively used conazoles, e.g. CYPRO, FLUS, PRO, and TEBU, was a result of altered steroido- genic gene expression in MA-10 cells. For that, MA-10 cells were exposed for 6 h to non-cytotoxic concentrations of the tested compounds based on preceding cytotoxicity exper- iments (data not shown). The five genes selected to be studied encode for the StAR protein, the main cholesterol transport carrier, as well as for the major enzymes involved
in the testis steroidogenesis route, i.e. Cyp11A1, Cyp17A1, 3ß-HSD1, and 17ß-HSD3. Exposure to cAMP (100 M) resulted in an increase in gene expression of StAR (49-fold), Cyp11A1 (6-fold), and Cyp17A1 (18-fold) in comparison with vehicle-treated cells (Fig. 2A). Gene expression of 3ß- HSD1 and 17ß-HSD3 did not significantly change upon cAMP treatment. Exposure to CYPRO, FLUS, PRO, or TEBU did not significantly affect gene expression of these five steroidogenic genes (Fig. 2A).
3.3. Expression of cholesterol biosynthesis genes
Effects of conazoles on cholesterol biosynthesis were studied since genes involved in “late” steroidogenesis (fol- lowing pregnenolone) could not explain the decrease in T synthesis. For proper T production, sufficient cholesterol is needed as steroid precursor. Gene expression of three enzymes involved in cholesterol biosynthesis was deter- mined, i.e. cytochrome P450 enzyme 51 (Cyp51), HMG-CoA reductase (HMG-CoA red), and cytochrome P450 oxidore- ductase (Por). Exposure to cAMP (100 µM) increased gene expression of Cyp51 (1.4-fold), HMG-CoA red (1.6-fold), and Por (1.4-fold) in comparison with vehicle-treated cells (Fig. 2B). Exposure to PRO increased the expression of
(% of time-matched DMSO control)
160
*
*
*
120
*
ROS formation
80
40
0
CYPRO FLUC
FLUS
HEXA
MYC PEN PRO TEBU TRIA TRIT
[100 μM]
Cyp51 (1.7-fold) and HMG-CoA red (1.9-fold) (Fig. 2B). Exposure to TEBU slightly decreased the expression of the Por gene (0.8-fold) (Fig. 2B). Other exposures did not change expression of the three cholesterol biosynthesis genes assessed (Fig. 2B).
3.4. Reactive oxygen species production
To further explore the nature of T secretion inhibition by MA-10 cells after exposure to certain conazole fungicides, we considered ROS formation as a possible cause for dete- rioration of Leydig cell function resulting in decreased T secretion. MA-10 cells were exposed to non-cytotoxic con- centrations (10 nM-100 µM) of the tested compounds. The control, rotenone, showed a ROS production of 158 ± 18% of the control at 48 h, indicating that the cells were able to pro- duce ROS. Only ROS levels in MA-10 cells exposed for 48 h to the highest concentration (100 µM) of FLUS, HEXA, PRO, and TEBU were statistically significantly increased com- pared to time-matched DMSO-treated control cells (Fig. 3). Increase in ROS formation was 1.2, 1.2, 1.3, and 1.4-fold compared with vehicle-treated control cells by FLUS, HEXA, PRO, and TEBU, respectively. The other tested conazoles (CYPRO, FLUC, MYC, PEN, TRIA, and TRIT) did not signifi- cantly change ROS production compared to vehicle-treated cells (Fig. 3). At lower concentrations (<100 }M) or at ear- lier time points (1 and 24 h) no differences in ROS formation between vehicle-treated and conazole-treated cells were observed (data not shown).
3.5. Inhibition of androgen receptor activation
Because AR activation is a prerequisite for proper sper- matogenesis, possible effects of conazole exposure on AR activation were determined using an AR reporter gene assay. T47D-ARE cells were exposed to non-cytotoxic con- centrations (10 pM to 100 (M) of the tested compounds. Testosterone (T) activated the AR in a concentration- dependent manner with an EC50 of 13.6 nM (Fig. 4A and Table 2). Exposure to conazoles alone did not significantly affect AR activation (data not shown). Next, cells were exposed to 20 nMT in combination with concentration ranges of the selected conazoles (10 pM to 100 }M) or the AR antagonist flutamide (FLUT; 10 nM to 100 (M). FLUT
| Compound | EC/IC50 [M] | BMR25%FLUT [M] | REP |
|---|---|---|---|
| T | 1.36E-08 | n.a. | n.a. |
| FLUT | 7.02E-06 | 1.98E-06 | 1.00 |
| CYPRO | 1.36E-05 | 5.25E-06 | 0.38 |
| FLUC | n.a. | n.a. | n.a. |
| FLUS | 1.19E-05 | 5.49E-07 | 3.61 |
| HEXA | 2.32E-05 | 7.64E-06 | 0.26 |
| MYC | 7.15E-05 | 7.06E-05 | 0.03 |
| PEN | 1.71E-05 | 5.54E-06 | 0.36 |
| PRO | 1.17E-05 | 9.43E-06 | 0.21 |
| TEBU | 2.55E-05 | 9.01E-06 | 0.22 |
| TRIA | 3.21E-05 | 1.60E-05 | 0.12 |
| TRIT | 1.07E-05 | 7.80E-06 | 0.25 |
concentration-dependently decreased AR activation with an IC50 value of 7.0 p.M (Fig. 4B and Table 2). All cona- zoles tested, except for FLUC, concentration-dependently inhibited T-induced AR activation with IC50s ranging from 10.7 to 71.5 MM (Fig. 4C-L and Table 2). All of the tested compounds inhibited T-induced AR activation by maxi- mally 82%.
In order to compare the potencies of the tested cona- zoles to inhibit AR activation, relative effect potencies (REPs) were calculated using the concentrations where inhibition of AR activation was similar to 25% inhibition by FLUT (BMR25%FLUT).
This leads to the following potency ranking based on the REP: FLUS>FLUT> CYPRO > PEN > HEXA >TRIT > TEBU > PRO > TRIA > MYC (Table 2).
4. Discussion
We show here that six of the ten tested conazole fungi- cides cause a decrease in basal T secretion in murine MA-10 Leydig cells. In addition, we demonstrated for two selected conazoles a concentration-dependent inhibition of LH- stimulated T secretion. These effects cannot be adequately explained by changes in steroidogenic and cholesterol biosynthesis gene expression nor by increased ROS
A
B
C
D
500-
150-
150
150-
RLU (% of control)
400
RLU (% of control)
RLU (% of control)
RLU (% of control)
100
100
H
100
300
HH
K
200
50
50
50
100
0
0
-10
-8
-6
4
9
8
-7
6
C
0
-14
-12
5
4
3
-12
-10
8
6
4
2
0
-12
-10
-6
-4
flutamide [log M]
-8
-2
testosterone [log M]
cyproconazole [log M]
fluconazole [log M]
E
F
G
H
150
150
150
150
RLU (% of control)
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RLU (% of control)
RLU (% of control)
100-
100.
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0
9
-8
-7
6
5
0
-4
3
-12
-10
8
-6
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2
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-10
8
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-2
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-10
-8
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-4
2
flusilazole [log M]
hexaconazole
[log M]
myclobutanil [log M]
penconazole [log M]
I
J
K
L
150-
150-
150
150
RLU (% of control)
RLU (% of control)
RLU (% of control)
RLU (% of control)
100
f
100
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·
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.9
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prochloraz [log M]
-2
-12
-10
-8
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tebuconazole [log M]
triadimefon [log M]
triticonazole [log M]
production. Further, nine of the ten tested conazoles inhibited T-induced AR activation in a reporter gene assay. These data show that some conazoles can act as anti- androgens via two modes of action. Anti-androgenic effects have been shown to cause adverse effects in both the adult as well as the developing male. Without adequate steroidogenesis leading to sufficient T secretion or by blockage of the AR response, spermatogenesis cannot be properly realized, resulting in abnormal or even absent sperm production and consequently sub- and/or infertil- ity of the adult male. In the fetus, proper T production and responsiveness are especially important during the masculinization programming window to ensure correct development into a phenotypical male [32].
In our study, five of the tested conazoles, i.e. FLUS, HEXA, PRO, TEBU, and TRIT, decreased basal T secre- tion by MA-10 cells by more than 50% compared with vehicle-treated control cells (Fig. 1A). FLUS and TEBU also concentration-dependently inhibited T secretion by MA- 10 cells stimulated with LH (Fig. 1B; REPSU10603 = 0.04 and 0.21, respectively), indicating that these compounds can act as (in vitro) T secretion inhibitors.
To mechanistically investigate the inhibition of T secretion by conazoles, we assessed gene expression of several enzymes involved in the “late” steroidogenic and
cholesterol biosynthesis pathways. However, the cona- zoles only inflicted minor effects on expression levels of the selected genes (Fig. 2), which cannot explain the inhibitory effects of these conazoles on T secretion in MA-10 Leydig cells. Makker et al. proposed a role for oxidative stress in the occurrence of male infertility [33]. However, levels of ROS resulting from exposure to conazoles were only mod- erately increased in our study (Fig. 3). Therefore, changes in ROS formation were also most likely not the cause of the decreased T secretion by MA-10 Leydig cells by the conazoles used in this study.
Our results are in accordance with the suggestion from earlier studies that conazoles show catalytic inhibition in the steroidogenic pathway [10,19,21]. Previous stud- ies indicate that conazoles have the potential to inhibit mammalian CYP51 enzyme activity via catalytic inhibi- tion [9,11]. CYP51 is highly expressed in the testis and has an important role in the cholesterol biosynthesis and ultimately in testosterone production [34,35]. T levels could also be affected by conazoles via catalytic inhibi- tion of other steroidogenic cytochrome P450 enzymes. Several studies have suggested that conazoles can tar- get cytochrome P450 enzymes, e.g. imidazoles also inhibit cytochrome P450 enzymes in human liver, thereby inhibi- ting hepatic T metabolism and lowering circulating levels,
and lymphoblast cells [20,36,37], This illustrates that cona- zoles are able to affect the function of multiple CYP enzymes, also the ones not specifically within the steroido- genic pathway, indicating that this type of enzymes could be one of the main targets for endocrine disruption by conazoles. We have previously shown that FLUS, ketocona- zole (an imidazole), and TEBU inhibit CYP17 activity in porcine adrenal cortex microsomes [20]. Furthermore, rat studies showed that hepatic cytochrome P450 activity is inhibited in vivo by FLUC and ketoconazole [36]. Previously, Kjaerstad et al. stated that the imidazoles are more potent inhibitors of T secretion by H295R cells than the triazoles [10]. This phenomenon was also seen for decreased T lev- els in fetal rat testes, where PRO showed higher potency than TEBU [38]. In contrast, our study did not show a stronger inhibition of T secretion in vitro by the imidaz- ole PRO compared to the triazole TEBU in murine MA-10 cells (Fig. 1A). In fact, TEBU is a quite potent inhibitor of T secretion in comparison with the CYP17 inhibitor SU10603 (REPSU10603 =0.21; Fig. 1B). Steroidogenesis in adrenal tis- sue occurs predominantly via DHEA whereas in the testes the main route for steroidogenesis includes production of androstenedione [39]. Since H295R cells are fetal-like adrenal cells and MA-10 cells are of testicular origin this might explain the differences in magnitude of response for conazoles to affect T secretion found between both cell sys- tems. The discordance seen between our in vitro results and the in vivo results might be explained by differential effects on aromatase (Cyp19) enzyme activity in the two systems used and a species difference of mouse Leydig cells versus rat fetal testes. It has been shown that MA-10 cells do not express the Cyp19 gene and estradiol does not repress Cyp17 and 3ßHSD gene expression [40]. In primary cultures of Leydig cells from C57BL/6j mice a significant upregulation of Cyp19 gene expression but coordinated suppression of the LHR, StAR, 3ßHSD, and Cyp17A1 genes was found, which was associated with attenuated andro- gen production compared to CBA/Lac mice [41]. Further, estradiol has been found to interfere with in vivo Leydig cell function in the rat, thereby lowering CYP17 activity leading to reduced T biosynthesis [42]. In vitro assays can be effectively used to study the effect of chemicals on a certain specific mechanism. The shortcoming of these assays is that they do not involve an intact organism, therefore lacking certain physiological feedback mecha- nisms within the body. A holistic evaluation of data from a panel of cell-based assays has shown to give a bet- ter prediction for the ranking of conazoles fungicides for in vivo toxicity data [38]. In addition, it has been shown previously that exposure to endocrine disruptors exerts differential effects on steroidogenesis in human, mouse, and rat testes, raising concern about the use of rodent models and extrapolation of results for human risk assess- ment [43]. Possibly, the species-difference in testicular responsiveness plays a role in the different potencies of prochloraz and TEBU in the rat developmental in vivo model described by Dreisig et al. and our in vitro mouse MA-10 study [38].
None of the conazoles showed AR agonistic activity but nine out of ten selected conazoles inhibited T-induced AR activation concentration-dependently in our reporter
gene assay (Fig. 4). The AR antagonistic activity of PRO and TEBU is in agreement with a previous study that used AR-transfected Chinese hamster ovary cells (CHO) [10]. In our present study with conazoles, FLUS is the most potent AR antagonist with an even higher potency (REP=3.61) than FLUT (REP = 1.00), a well-studied pharmacological AR antagonist. All of the selected conazoles contain at least one hexacyclic moiety (Table 1), which may have a func- tion comparable to the hexacyclic moiety of T, and thus may cause a competitive receptor binding between these fungicides and androgens [44]. Comparison of the chemi- cal structures of these selected conazoles (Table 1) shows that FLUS is the only conazole containing an additional hexacyclic moiety. Possibly, this extra moiety plays a role in the AR antagonistic properties of FLUS. Further studies on receptor-ligand kinetics are needed to determine the nature of these antagonistic interactions.
Besides environmental exposure, individuals can also be exposed to conazoles via pharmaceutical application. FLUC has a pharmacotherapeutic application and treated patients showed serum levels ranging from 16.3 to 25.8 p.M after oral administration of 200 mg FLUC per day [45]. It should be noted that these blood levels are higher than the maximum medium concentration of 10 µM that caused an inhibition of T secretion by MA-10 cells in this study. More- over, the fast uptake rate with a Tmax of approximately 2 h together with the plasma half-life of approximately 30 h in humans suggests that significant internal exposure to conazoles can occur, possibly even resulting in accumu- lation of these compounds leaving more opportunity for causing (adverse) effects, e.g. inhibition of CYP enzymes [46].
In addition, because different crops are treated with different fungicides and conazole mixtures are commer- cially available, the possibility exists that people may be frequently exposed to several conazoles simultaneously. Mixtures of individual endocrine active compounds, including conazoles, have been shown to cause additive effects and antagonism has also been observed [47]. Also combinations of low doses of multiple conazoles have been shown to cause additive effects [48]. An earlier study by Kjaerstad et al. showed additivity of a mixture containing two triazoles (propiconazole and TEBU) and one imidaz- ole (epoxiconazole) on AR antagonism in AR-transfected CHO cells as well as T synthesis in H295R cells [49]. Like- wise, the triazole propiconazole in combination with other pesticides showed additive AR activity antagonism [50]. Hence, low effect concentrations of conazoles, to which humans are most likely frequently exposed and in mix- tures, may potentially pose a risk for endocrine disruptive effects. Unfortunately, a proper risk assessment is ham- pered by the lack of (systemic) human exposure data.
5. Conclusion
In summary, this in vitro study shows clear anti- androgenic effects of several conazole fungicides. These anti-androgenic effects suggest that potential testicular toxicity can arise from two mechanisms: inhibition of T secretion and AR antagonism. In view of the dual anti- androgenic effects of the conazoles described here, further
studies on the male reprotoxic effects of conazole fungi- cides in combination with accurate exposure data are highly recommended.
Transparency document
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Acknowledgements
We gratefully acknowledge Ing. Sandra M. Nijmei- jer for the excellent technical assistance. This research was funded by the European Community’s Seventh Framework Programme ([FP7/2010-2014]; GA244236), ChemScreen (http://www.bds.nl/chemscreen; van der Burg et al., 2011), and the Doerenkamp-Zbinden Founda- tion (http://www.doerenkamp.ch).
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