ELSEVIER
Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
Toxicology Letters
TL
An in vitro investigation of endocrine disrupting effects of trichothecenes deoxynivalenol (DON), T-2 and HT-2 toxins
D.G. Ndossia,b,*,1, C. Frizzellc,1, N.H. Tremoenª, C.K. Fæsted, S. Verhaegenª, E. Dahla, G.S. Eriksend, M. Sørlie e, L. Connollyc, E. Ropstad ª
a Norwegian School of Veterinary Science, Oslo, Norway
b Sokoine University of Agriculture, Morogoro, Tanzania
” Institute of Agri-food and Land Use, School of Biological Sciences, Queen’s University Belfast, Northern Ireland, United Kingdom
d Norwegian Veterinary Institute, Oslo, Norway
e Norwegian University of Life Sciences, Norway
HIGHLIGHTS
H295R cells and RGA cells were exposed with DON, T-2 and HT-2 toxins.
We observed reduced cell viability with increasing toxin concentration.
Increased toxin concentration was associated with reduced hormone production.
There was neither agonistic nor antagonistic effects following reporter gene assays.
Trichothecenes modulated the expression of most of the steroidogenic genes analyzed.
ARTICLE INFO
Article history: Received 20 March 2012 Received in revised form 5 September 2012 Accepted 5 September 2012 Available online 12 September 2012
Keywords: Endocrine disruptor Steroidogenesis H295R
Reporter gene assay Gene expression
ABSTRACT
Trichothecenes are a large family of chemically related mycotoxins. Deoxynivalenol (DON), T-2 and HT-2 toxins belong to this family and are produced by various species of Fusarium. The H295R steroidogenesis assay, regulation of steroidogenic gene expression and reporter gene assays (RGAs) for the detection of androgen, estrogen, progestagen and glucocorticoid (ant)agonist responses, have been used to assess the endocrine disrupting activity of DON, T-2 and HT-2 toxins.
H295R cells were used as a model for steroidogenesis and gene expression studies and exposed with either DON (0.1-1000 ng/ml), T-2 toxin (0.0005-5 ng/ml) or HT-2 toxin (0.005-50 ng/ml) for 48 h. We observed a reduction in hormone levels in media of exposed cells following radioimmunoassay. Cell via- bility was determined by four colorimetric assays and we observed reduced cell viability with increasing toxin concentrations partly explaining the significant reduction in hormone levels at the highest toxin concentration of all three trichothecenes.
Thirteen of the 16 steroidogenic genes analyzed by quantitative real time PCR (RT-qPCR) were signifi- cantly regulated (P<0.05) by DON (100 ng/ml), T-2 toxin (0.5 ng/ml) and HT-2 toxin (5 ng/ml) compared to the control, with reference genes (B2M, ATP5B and ACTB). Whereas HMGR and CYP19 were down- regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. DON further up-regulated CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulated NR5A1. T-2 toxin caused down-regulation of NR0B1 and NR5A1 whereas HT-2 toxin induced up-regulation of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not significantly affected by any of the trichothecenes in the present study.
Although the results indicate that there is no evidence to suggest that DON, T-2 and HT-2 toxins directly interact with the steroid hormone receptors to cause endocrine disruption, the present findings indicate that exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, steroidogenesis and alteration in gene expression indicating their potential as endocrine disruptors.
@ 2012 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author at: Norwegian School of Veterinary Sciences, Department of Production Animals Medicine, Post box 8146 Dep, 0033 Oslo, Norway.
E-mail address: doreen.ndossi@nvh.no (D.G. Ndossi).
1 Contributed equally to this work.
1. Introduction
Trichothecenes are a group of structurally related mycotoxins produced by a range of fungi, with Fusarium species as the main producers worldwide (Scott, 1989). F. graminearum and F. culmorum are important for the production of deoxynivalenol (DON) (WHO, 1990), whereas F. sporotrichioides, F. poae and F. langsethiae are responsible for the production of T-2 and HT-2 toxins (Logrieco et al., 1992; Torp and Langseth, 1999). The extent of infection depends on pre-harvest conditions on the field. The toxin produc- tion may occasionally continue even post-harvest until the grain is sufficiently dry. Conditions are species-dependent, and gener- ally little is known about F. langsethiae. Trichothecenes may be found on cereals such as wheat, maize, barley, corn, oats and rye as well as in processed grains because of the extreme sta- bility of these compounds (Canady et al., 2001; Placinta et al., 1999; Scott, 1989). Various food and food raw materials have been analyzed for the occurrence of trichothecenes. DON is the most commonly detected trichothecene in cereal grains (Canady et al., 2001). Hydrolysis and deacetylation of trichothecenes is com- monly found in animal systems. T-2 is rapidly metabolized to HT-2 toxin in vivo. Glucuronidation is also an important biochemical pathway for the metabolism of DON, T-2 and HT-2 toxins (He et al., 2010).
Trichothecenes are responsible for a wide variety of toxic effects in animals. The concentration of the mycotoxin, duration of expo- sure and species involved can all have an impact on the toxicity. Differences in their toxicity are due to their chemical structure (Ueno, 1985). T-2 toxin is the most acutely toxic trichothecene pro- duced by Fusarium fungi (Hanelt et al., 1994). The general toxicity, haematoxicity and immunotoxicity of T-2 and HT-2 toxins are of critical concern. DON can induce vomiting, cause feed refusal and reduce growth rates and feed conversion. Feeding studies indicate that pigs are the most sensitive to these toxins, in comparison to other farm animals (Eriksen and Pettersson, 2004).
In humans trichothecenes have been associated with several toxicological effects, including disease outbreaks. DON has been implicated in outbreaks of human gastrointestinal disorders in China, India and South Africa (Bhat et al., 1989; Luo, 1988) while T-2 toxin was reported in the outbreaks of fatal Alimentary Toxic Aleukia (ATA) in the Soviet Union (Joffe, 1986). Trichothecenes are reported as protein synthesis inhibitors binding to 60s ribosomal subunit and interact with the enzyme peptidyltransferase lead- ing to inhibition of peptide bond formation (Cundliffe and Davies, 1977). DON and T-2 toxin are reported to inhibit cell proliferation, RNA and DNA synthesis, and induce apoptosis (Nagase et al., 2001; Rotter et al., 1996) partly due to inhibition of protein synthesis. DON and T-2 toxins cause immunomodulatory effects varying from sup- pression to stimulation of the immune system (Bondy and Pestka, 2000; Thuvander et al., 1999).
The potential of these trichothecenes to act as endocrine dis- ruptors has been the subject of more recent research and continues to be investigated. DON has been implicated in impairing repro- ductive performance in pigs, due to its ability to inhibit oocyte maturation (Alm et al., 2002, 2006), reduce feed intake, and impair oocyte and embryo development (Tiemann and Danicke, 2007). It has also been shown that DON has dose-dependent effects on steroid hormone production by porcine granulosa cells (Ranzenigo et al., 2008) inhibiting IGF-I-induced progesterone production and FSH plus IGF-I-induced Cyp19A1 and Cyp11A1 mRNA abundance at higher doses (100-1000 ng/ml). However, another study look- ing at progesterone release by ovarian granulosa cells found that at similar high doses of DON (1000 ng/ml) progesterone release was stimulated (Medvedova et al., 2011). The effect of DON on the male reproductive system has been studied in mice. Serum testos- terone concentrations were observed to decrease in a dose-related
manner, but the exact mechanism was not identified (Sprando et al., 2005).
Similar to DON, the potential impact of T-2 toxin on reproductive performance in pigs has been investigated by looking at the effects of this mycotoxin on steroid hormone production in porcine gran- ulosa cells (Caloni et al., 2009). The T-2 toxin had potent inhibitory effects on progesterone and to a greater extent estradiol produc- tion, with T-2 being much more potent at inhibiting steroidogenesis than DON. T-2 toxin is reported to inhibit testosterone production in gerbil testicular interstitial cells (Fenske and Fink-Gremmels, 1990). Animal studies have shown abnormal reproductive effects of T-2 toxin including reduced testosterone concentration in male rabbits (Kovacs et al., 2011), reduced sperm production in male mice (Yang et al., 2010) and delayed follicle maturation and ovula- tion in heifers and ewes (Huszenicza et al., 2000).
An endocrine disruptor is defined as an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organ- ism, or its progeny, or (sub)populations. A potential endocrine disruptor is an exogenous substance or mixture that possesses properties that might be expected to lead to endocrine disrup- tion in an intact organism, or its progeny, or (sub)populations (WHO/IPCS, 2002). In vitro bioassays are useful for evaluating spe- cific toxicological actions of chemicals. In vitro systems have been established to evaluate endocrine disrupting compounds whose activity is via hormone receptors and/or steroidogenesis pathways (Connolly et al., 2011). Reporter gene assays (RGAs) and the H295R steroidogenesis assays have previously been employed to detect the endocrine disrupting potential of zearalenone and its metabo- lites (Frizzell et al., 2011). Endocrine disrupting effects of toxins and chemicals are increasingly being studied using human adrenocor- tical carcinoma cells, H295R (Hecker and Giesy, 2008). The H295R cells physiology resembles that of zonally undifferentiated human foetal adrenal cells and the cells have the steroidogenic ability simi- lar to the three zones of the adult adrenal cortex (Gazdar et al., 1990; Staels et al., 1993). These cells also express both alpha and beta estrogen receptors (Somjen et al., 2003; Montanaro et al., 2005) as well as androgen receptors (Rossi et al., 1998). Measurement of gene expression in the H295R cell model has been demonstrated as a useful means to evaluate the potential of chemicals to interfere with the expression of steroidogenic enzymes and also provide a means of profiling the mode of action of chemicals (Gracia et al., 2006; Hilscherova et al., 2004; Zhang et al., 2005).
DON and T-2 have been shown to act as potential endocrine disruptors independent of steroid hormone receptors, yet no infor- mation is available on the interaction of the DON, T-2 and HT-2 toxins on the steroid hormone receptors. The aim of this study was to determine any potential endocrine disrupting effects of the trichothecenes DON, T-2 and HT-2 toxins, at the level of steroid hormone synthesis, steroidogenic genes expression and nuclear receptor transcriptional activity, using estrogen, androgen, pro- gestagen and glucocorticoid RGAs.
2. Materials and methods
2.1. Chemicals
The mycotoxins DON, T-2 and HT-2 and the hormones 170-estradiol (E2), testosterone (T2), progesterone (P4) and hydrocortisone (H) were obtained from Sigma-Aldrich (St Louis, MO, USA) and dissolved in methanol (Sigma-Aldrich, St Louis, MO, USA) at a final concentration of 0.1%, v/v or 0.5% in media for the H295R steroidogenesis and RGA respectively.
2.2. Cell culture
2.2.1. H295R cell model
The H295R human adrenocortical carcinoma cells were obtained from the Amer- ican Type Culture Collection (ATCC # CRL-2128, ATCC Manassas, VA, USA) and cultured as previously described (Hilscherova et al., 2004). Briefly the cells were
grown in 75 cm2 flasks with 12.5 ml of supplemented medium at 37℃ and 5% CO2 atmosphere. The medium used was 1:1 mixture of Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F-12 Nutrient (Gibco, Invitrogen, Paisley, UK) con- taining 15 mM HEPES buffer and further supplemented with 1% ITS+ Premix (BD Biosciences, Bedford, MA, USA), and 2.5% NuSerum (BD Biosciences). The medium was changed two to three times a week and cells were detached from flasks for sub-culturing using 0.25% trypsin/0.53 nM EDTA (Gibco, Invitrogen, Paisley, UK) at a confluence of 80-90%.
2.2.2. Reporter gene assays
A panel of reporter gene assays (RGAs) were developed from human mammary gland cell lines by transformation with the luciferase gene under the control of a steroid hormone inducible promoter (Willemsen et al., 2004). The MMV-Luc cell line is specific for the detection of estrogens, TARM-Luc for androgens and progestagens, TM-Luc for progestagens and TGRM-Luc for glucocorticoids and progestagens. All cell lines were cultured in 75 cm2 tissue culture flasks (BD Biosciences, Bedford, MA, US) at 37 ℃ with 5% CO2 and 95% humidity. All RGA cell culture reagents were supplied by Invitrogen, Paisley, UK. The cells were routinely grown in cell culture medium containing Dulbecco’s Modified Eagle Medium (DMEM), 10% foetal bovine serum and 1% penicillin streptomycin. DMEM without phenol red was used when culturing the estrogen responsive MMV-Luc cell line. Cells were transferred at least two passages prior to RGA analysis into assay medium (DMEM with 10% hormone depleted serum).
2.3. Treatment of H295R cells
All experiments were conducted in 24-well cell culture plates (Primaria Multiwell™ Becton Dickinson and Co., NJ, USA). Cell suspension at a concentra- tion of 3 x 105 cells/ml was added to each well and the plates incubated at 37 ℃, 5% CO2 for 24h to allow the H295R cells to attach. After the attachment period the medium was changed and the cells were exposed for 48 h with either DON (0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml (0.34 nM, 3.38 nM, 33.75 nM, 0.34 µ.M, 3.38 µM respectively)), T-2 toxin (0.0005 ng/ml, 0.005 ng/ml, 0.05 ng/ml, 0.5 ng/ml, 5 ng/ml (1.07 pM, 10.73 pM, 0.11 nM, 1.07 nM, 10.73 nM respectively)) or HT-2 toxin (0.005 ng/ml, 0.05 ng/ml, 0.5 ng/ml, 5 ng/ml, 50 ng/ml (11.79 pM, 117.92 pM, 1.18 nM, 11.79 nM, 117.92 nM respectively)). Forskolin 10 p.M was used as a positive control, 0.1% methanol as solvent control and some cells left unex- posed (blank medium control). Assays for each concentration were carried out in triplicate and each experiment was repeated three times. At the end of each exper- iment, medium from each well was transferred into labelled 2 ml plastic vials and stored at -20℃ until hormone analysis.
2.4. Reporter gene assay
Cells were seeded at a concentration of 4 x 105 cells/ml, 100 pl/well, into white walled 96 well plates with clear flat bottoms (Greiner Bio-One, Frickenhausen, Germany) and the cells were allowed to attach for 24h. The following day, stan- dards and samples were prepared 1:100 v/v by adding 10 ul of the relevant steroid hormone or standard to 1 ml of assay media, and without removing the media that the cells were seeded in, 100 ul of this dilution of standard or sample was added to the plate in triplicate giving a final methanol concentration of 0.5% in the wells. The following standards were used with each cell line: 17ß-estradiol (1.36 ng/ml) for the MMV-Luc cell line, testosterone (14.4 ng/ml) for the TARM-Luc cell line, proges- terone (157 ng/ml) for the TM-Luc cell line and hydrocortisone (181 ng/ml) for the TGRM-Luc cell line. Media spiked with DON (0.025-2500 ng/ml (0.084 nM-8.4 p.M)), T-2 (0.023-23.3 ng/ml (0.05-50nM)) and HT-2 (0.005-50 ng/ml (11.8 pM-118 nM)) were added for the agonist test. Antagonist tests were carried out by incubating these concentrations of DON, T-2 and HT-2 toxins with 1.36 ng/ml 17ß-estradiol (MMV-Luc), 14.4 ng/ml testosterone (TARM-Luc), 157 ng/ml progesterone (TM-Luc) and 181 ng/ml hydrocortisone (TGRM-Luc). Once the standards/samples were added the cells were incubated for 24 h (MMV-Luc cell line) or 48 h (for all other cell lines). After incubation, the supernatant was discarded and the cells washed twice with phosphate buffered saline (PBS) prior to lyses with 20 ul cell culture lysis buffer (Promega, Southampton, UK). Finally, 100 ul luciferase (Promega, Southampton, UK) was injected into each well and chemiluminesence measured using the Mithras Multimode Reader (Berthold, Germany). All experiments were repeated in 3 inde- pendent exposures. The responses of the standards or samples were compared with the negative control (0.5% methanol in media).
2.5. Determination of cell viability
2.5.1. H295R cell line
2.5.1.1. AlamarBlue assay. Briefly, cells were seeded at a concentration of 3 x 105 cells/ml for 24h and then exposed for 48 h with the different concentra- tions of mycotoxins and the controls. After removing the supernatant 1 ml fresh medium containing 10% AlamarBlue (Invitrogen, Carlsbad, CA, USA) was added into each well and incubated for 3 h at 37 ℃ and 5% CO2. A sample of 100 ul was then collected from each well into a 96-well plate (Thermo Fisher Scientific, Roskilde, Denmark) and the absorbance measured at 570 nm and 600 nm in a VICTOR3™M
spectrophotometer (Perkin Elmer, Shelton, USA). Cell viability was expressed as a % of solvent control.
2.5.1.2. Neutral red uptake. The neutral red assay (NR) is based on the ability of viable cells to incorporate the supravital dye, NR into their lysosomes after incu- bation with toxic agents (Babich and Borenfreund, 1991; Repetto et al., 2008). The assay was performed as described by Repetto et al. (2008) with minor modifica- tion. Briefly, the cells were seeded in the 60 inner wells of 96-well-plates (Nunc, Roskilde, Denmark) at a concentration of 5 x 104 cells/well and incubated overnight at 37℃ and 5% CO2. The outer wells were filled with 200 ul medium. Cells were exposed in fresh medium containing the different concentrations of trichothecenes and the solvent control (0.1% MeOH) for 48 h. The supernatant was discarded and the cells incubated for 2 h at 37 ℃ with 100 pl/well of pre-incubated medium con- taining 40 µg/ml NR prepared from stock solution of 4 mg/ml NR (Sigma-Aldrich), dissolved in PBS. The NR medium was then discarded and the cells washed with PBS before extracting the dye by adding NR destaining solution (acidified ethanol) made from 50% ethanol 96% (Kemetyl, Norge), 49% deionized water and 1% glacial acetic acid (Merck, Darmstadt, Germany). After shaking the plate on a microtiter plate shaker for 10 min, the optical density of the extracted dye was measured at 540 nm in Victor2 Multilabel Counter (Wallac) using blanks which contain no cells as a reference. Control cells treated with 0.1% MeOH represented 100% viability. The experiment was repeated three times and dose-response curves plotted after converting the mean data values to percentages of the control response.
2.5.1.3. BrdU assay. A cell proliferation kit (Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 11647229001) was used to determine the amount of BrdU incorporated in the DNA. This calorimetric immunoassay kit is a non-radioactive alternative to the 3H-thymidine incorporation assay. The assay was performed according to manufacturer’s instructions. Briefly, 200 ul cell suspension was added into the 60 inner well of 96-well-plates (Nunc, Roskilde, Denmark) for seeding cells at a concentration of 1 x 104 cells/well and incubated overnight at 37 ℃ and 5% CO2. The outer wells were filled with 200 pl PBS. Cells were exposed in fresh medium con- taining the different mycotoxins at 5 different concentrations including the solvent control (0.1% MeOH) for 33 h before labelling the cells with 10 p.M BrdU and further incubated for 15 h. The cells were fixed and the plates treated with blocking buffer (gelatin) before addition of the anti-BrdU-POD (monoclonal antibody from mouse conjugated with peroxide) which binds to the BrdU incorporated into the newly synthesized DNA. The immune complex was detected by the substrate reaction (tetramethylbenzidine) and the reaction stopped with 1 M H2SO4. The absorbance of the yellow reaction product was measured with VICTOR3™ (Perkin Elmer, Shel- ton, USA) at 450 nm and 690 nm as a reference wavelength. The experiment was repeated three times and the dose-response curves plotted after converting the mean data values to percentages of the control response.
2.5.2. RGA cell lines
2.5.2.1. MTT assay. Cells were seeded as for the RGA’s and DON (0.025-2500 ng/ml (0.084 nM-8.4p.M)), T-2 (0.023-23.3 ng/ml (0.05-50nM)) and HT-2 (0.005-50 ng/ml (11.8 pM-118 nM)) added to the cells. The plates were incu- bated with the standards in triplicate as for the RGAs. The supernatant was then discarded and the cells washed once with PBS. A 50 ul volume of thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich, St Louis, MO, USA) solution (5 mg/ml stock in PBS diluted 1:2.5 in assay media) was added to each well and the cells incubated for 3h. Viable cells convert the soluble yellow tetrazolium salt MTT to insoluble purple formazan crystals by the action of mitochondrial succinate dehydrogenase. The liquid was removed from the wells and 200 ul of DMSO added to dissolve the crystals. The plate was incubated for 10 min at 37℃ with agitation before measuring the optical density at 570 nm with a reference filter at 630 nm. Samples were analyzed in 3 independent assays. Viability was calculated as the % absorbance of the sample when compared with the absorbance of the control (0.5% methanol in media).
2.6. Hormone quantification
Frozen media samples from the 48 h exposed cells were thawed at room tem- perature prior to analysis. The levels of estradiol, testosterone and cortisol were quantified in duplicate by solid phase radioimmunoassay Coat-a-Count RIA kits (Diagnostic Products Corporation, Los Angeles, USA). Progesterone hormone was measured by Spectria Progesterone RIA kits (Orion Diagnostica, Espoo Finland). All kits were used according to manufacturer’s instructions except for the standards that were prepared in cell culture medium rather than the supplied standards.
2.7. RNA isolation
Cells were seeded in 24 well plates at a concentration of 3 x 105 cells/ml/well and incubated overnight for attachment at 37 ℃, 5% CO2 and 95% air. The cells were then exposed in fresh media for 48 h with either 100 ng/ml (0.34 p.M) DON; 0.5 ng/ml (1.07 nM) T-2 toxin; 5 ng/ml (11.79 nM) HT-2 toxin; or 0.1% MeOH (Solvent control) in triplicate wells. The plates were snap-frozen and stored at -75 ℃ for RNA iso- lation at a later stage. Five separate experiments were performed. Total RNA was
| Gene | Enzyme name | Function |
|---|---|---|
| M2CR (ACTHR) | Melanocortin 2 receptor (adenocorticotropin hormone receptor) | This receptor is mediated by G proteins which activate adenylate cyclase The melanorcortins are involved in several physiological functions including steroidogenesis |
| HMGR (HMGCR) | Hydroxy-methyl-glutaryl CoA reductase | Rate-limiting enzyme of cholesterol biosynthesis, reduction of HMG CoA into CoA and mevalonic acid |
| StAR | Steroidogenic acute regulatory protein | Key role in steroid hormone synthesis, mediates cholesterol transfer to the inner mitochondrial membrane |
| CYP11A (Cholesterol scc) | Cytochrome P450 11A | Catalyzes the first and rate limiting enzymatic step in biosynthesis of steroid hormones; cholesterol side-chain cleavage to form pregnenolone |
| CYP11B1 | Cytochrome P450 11B1 | Conversion of 11-deoxycortisol to cortisol |
| CYP11B2 | Aldosterone synthetase | Catalyzes the 11- and 18-hydroxylation; and 18-oxidation |
| CYP17 | Cytochrome P450 17 | Conversion of pregnenolone and progesterone to their 17-alpha-hydroxylated products |
| CYP21 | Cytochrome P450 21 | Catalyzes the 21-hydroxylation reactions of steroids. Adrenal synthesis of mineralocorticoids and glucocorticoids |
| HSD3B2 | Beta-hydroxysteroid dehydrogenase 2 | Catalyzes the oxidative conversion of delta(5)-ene-3-beta-hydroxy steroid, and the oxidative conversion of ketosteroids Important in the biosynthesis of all steroid hormones |
| HSD17B1 | 17ß hydroxysteroid dehydrogenase 1 (17 ketoreductase) | NAD(H)- and/or NADP(H)-dependent enzymes that catalyze the oxidation and reduction of 17-hydroxy- and 17-ketosteroids respectively |
| HSD17B4 | 17ß hydroxysteroid dehydrogenase 4 | Peroximal beta-oxidation pathway for fatty acids Catalyzes the formation of 3-ketoacyl-CoA intermediates |
| CYP19 | Aromatase | Conversion of androgen to estrogen |
| CYP1A1 | Cytochrome P450 1 A1 | Catalyzes many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids |
| NR5A1 (SF1) | Nuclear receptor 5A1 | Transcriptional activator for e.g. CYP11A, CYP11B, CYP21B and StAR Essential for sexual differentiation and formation of the primary steroidogenic tissues |
| NR0B1 | Nuclear receptor 0B1 | Development of the hypothalamic-pituitary-adrenal-gonadal axis Acts as a coregulatory protein inhibiting transcriptional activity of other nuclear receptors |
| EPHX | (Microsomal) epoxide hydrolase | Metabolism of endogenous and exogenous compounds in oxidative stress |
isolated using Qiagen RNeasy mini-kit (Qiagen, Crawley, UK) following manufac- ture’s instruction. Briefly, cells were lysed in the culture plate with addition of lysis buffer to each well and cells scraped with pipette tip and mixed well by pipetting up and down. Cells were homogenized in QIAshredder spin column by centrifuging for 2 min at 13,000 rpm before RNA extraction in RNeasy spin column. Residual DNA was removed by addition of RNase-free-DNase I mixture (RNase-Free DNase Set, Qiagen) and incubation for 10 min at room temperature. After the final wash, the spin columns were transferred into fresh RNase free 1.5 ml microcentrifuge tubes and RNA eluted by adding RNase-free H2O directly onto the fibre matrix at the cen- tre of the spin column and centrifuged for 1 min at 13,000 rpm. The RNA sample was stored at -75℃. The quantity of RNA was determined by NanoDrop technique (Thermo-Scientific, Waltham, MA, USA) and the quality determined on nano chips by Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
2.8. Gene expression
The most stable reference genes were predicted by the GeNorm human detec- tion kit and software (PrimerDesign Ltd., Southampton, UK) and NormFinder (Molecular Diagnostic Laboratory, Aarhus University Hospital, Denmark). Six genes (B2M, ACTB, EIF4A2, YWHAZ, ATP5B, GAPDH) were tested for stability in our cells. Whereas B2M was the most stable gene, ATP5B and ACTB gave the best stable com- bination of genes and these three were selected as reference genes in this study.
Table 1 shows a list of genes that were analyzed in this study. Primer sequences for CYP11A, CYP11B2, CYP17, CYP19, CYP21, 3BHSD2, 17BHSD1, 17BHSD4, StAR, ACTB and HMGR were obtained from Hilscherova et al. (2004), EPHX was obtained from Mollerup et al. (2006) while primer sequences for CYP1A1, CYP11B1, MC2R, NR5A1, NR0B1, GAPDH and YWHAZ were obtained from Zimmer et al. (2011). All primers were synthesized by Sigma-Aldrich (St. Louis, MO, USA) except for CYP11B1 that was manufactured by PrimerDesign Ltd (Southampton, UK). Speci- ficities for all primer pairs were checked using nucleotide BLAST and primer BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The primers were used at a working con- centration of 10 p.M. Prior to analysis, the primers annealing temperature and cDNA concentration were checked.
cDNA synthesis and quantitative reverse transcriptase PCR was performed using Superscript Platinum III Two-Step qRT-PCR with SYBR Green (Invitrogen) accord- ing to manufacturer’s instructions. Synthesis of cDNA was done in Peltier Thermal Cycler-225 (MJ Research, Waltham, MA, USA) and RT-qPCR was performed by a DNA engine Thermal Cycler with Chromo 4 Real-Time Detector (MJ Research) and its
software, Opticon Monitor 3, (Bio-Rad Laboratories, Hercules, CA, USA) as described previously (Zimmer et al., 2011). Briefly, RNA samples were diluted and technically duplicated before synthesis of cDNA. Negative controls without reverse transcrip- tase and negative template controls were included in the reverse transcription step. The synthesized cDNA was further diluted and a total of 7.5 ng cDNA (assuming full reverse transcriptase efficiency) was added to the qPCR reaction. The cycles were at 50℃, 2 min (UDG incubation); 95 ℃, 2 min (enzyme activation) followed by 40 cycles of 95 ℃ for 15s, 62 ℃ for 30s and 72 ℃ for 30s. Primer-dimer genomic DNA and other DNA contaminations were monitored during the experiment by including the melting curve (65-95 ℃ and read every 0.3 ℃ for 1 s) at the end of each run.
2.9. qPCR data analysis
Data from the Opticon Monitor 3 software were transferred into Excel spread sheets for processing and analysis. The ACt was calculated from the difference in expression between the gene of interest and the mean expression of the three reference genes. The AACt was calculated from the difference in ACt between cells exposed to solvent control and the mycotoxins in subject. Fold change was calculated using 2-AACt as described by Livak and Schmittgen (2001).
2.10. Statistical analysis
Data was analyzed by JMP 9 software (SAS Institute Inc., Cary, NC, USA). All data were expressed as mean ± standard error of the mean of triplicate indepen- dent experiments. A one way analysis of variance, ANOVA with Student’s t-test was used to determine significance differences between treated and the corresponding controls. A P-value of <0.05 was considered significant. Differences in mean hor- mone concentrations in exposed cells were compared to the solvent control using Dunnet’s test where the mean concentrations were tested for significance differ- ence at the 95% confidence level. Cell viability differences between exposed cells and solvent control were analyzed using Dunnet’s test as well.
For RGAs, dose-response curves were fitted with SlideWrite Plus V6 software using the sigmoidal dose-response curve equation, Y=Bottom + (Top - Bottom)/(1 + 10(Log EC50-X)), here X is the logarithm of con- centration, Y the response, and Bottom and Top are fixed to 0% and 100% respectively of the maximum response of the standard used in each test. Fold induction was measured by calculating the ratio of a response when compared with
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50
1
10
DON concentration (ng/ml)
T-2 toxin concentration (ng/ml)
HT-2 toxin concentration (ng/ml)
the negative control (n-fold). All values shown are expressed as mean ± standard deviation.
3. Results
3.1. Viability and cytotoxicity
In H295R cells, viability was assessed by AlamarBlue assay, neu- tral red uptake (NR assay) and BrdU incorporation following 48 h exposure of cells to trichothecenes. We observed reduced cell via- bility that was dose dependent in all three assays. Except for the NR assay results for HT-2 toxin at the highest concentration, all the three assays showed the same results in all concentrations used in the present study (Fig. 1). Exposure to the highest concentra- tions, 1000 ng/ml DON, 5 ng/ml T-2 toxin and 50 ng/ml HT-2 toxin led to a significant reduction in viability compared to untreated cells (P<0.05). According to the NR assay, HT-2 toxin showed increased cytotoxicity at 50 ng/ml (117.92 nM) (only about 37% viable cells). Inhibition of DNA synthesis has been demonstrated by dose response curves obtained using the BrdU bioassay, especially in the highest concentrations of trichothecenes tested. One higher concentration of T-2 toxin, 50 ng/ml, was included only in the BrdU assay and this resulted to a clear dose response curve (Fig. 1).
Cytotoxicity was observed in all RGA cell lines at the highest mycotoxin concentrations tested (2500 ng/ml DON, 23.3 ng/ml T-2
and 50 ng/ml HT-2) (Fig. 2). A change in cell morphology and some reduction in viability at the 1/10 dilution of these concentrations was also observed, however to a lesser extent.
3.2. Effect on hormone production
Hormone production levels in H295R cells were determined by RIA after 48 h exposure. The intraassay and interassay coefficients of variations were less than 10%. 10 µM forskolin that was used as a positive control, induced steroidogenesis in our cells, where all hormone levels were increased compared to the control cells. Estra- diol concentration for instance increased about 30 folds compared to that of untreated cells (results not shown). Basal productions of all hormones were not significantly affected by low doses of mycotoxin but there was a trend towards inhibition of the pro- duction with increased toxin dosage. The production of estradiol, testosterone and cortisol hormones were reduced with increased DON concentration. Significant inhibition of estradiol and testos- terone hormone production was observed following exposure to ≥100 ng/ml (P<0.05) where estradiol decreased to below detec- tion limit in cells exposed to 1000 ng/ml DON. Testosterone levels in cells exposed to higher concentrations of DON (100-1000 ng/ml) were lower than in the control cells. There was a significant increase in progesterone production (P<0.0001) in cells exposed to 1000 ng/ml (Fig. 3).
Cell Viability of RGA Cell Lines following exposure to DON
Cell Viability of RGA Cell Lines following exposure to T-2 Toxin
Cell Viability of RGA Cell Lines following exposure to HT-2 Toxin
120-
+ AR Cell Line
110-
100
+ AR Cell Line
120-
110
110
+ AR Cell Line
100
-+- ER Cell Line
90
+- ER Cell Line
100
+ ER Cell Line
Cell Viability (% )
90
+ PR Cell Line
Cell Viability (% )
80
Cell Viability (% )
80-
+ PR Cell Line
90
70-
80-
+ PR Cell Line
70-
+- GR Cell Line
-+- GR Cell Line
70-
+ GR Cell Line
60-
60-
50-
50-
60
50-
40-
40-
30-
40-
30-
20-
30-
20-
20-
10
10.
10
0
0
0
0.025
0.25
2.5
25
250
2500
0.023
1.23
2.33
23.3
0.005
0.05
0.5
5
50
DON (ng/ml)
T-2 (ng/mL)
HT-2 (ng/ml)
At 0.0005 ng/ml T-2 toxin concentration, basal production of estradiol was slightly below that of the control cells and tended to increase with increasing toxin concentration to 0.05 ng/ml. Estra- diol levels were significantly reduced (P<0.0001) in cells exposed to 5 ng/ml T-2 toxin by about 2.5 folds less than in control cells. There was no significant change in progesterone production at lower T-2 toxin concentrations (0.0005-0.05 ng/ml) but the level increased significantly in cells exposed to 5 ng/ml. Testosterone production was dose-dependently inhibited (P<0.05) whereas the production of cortisol was significantly reduced only with the high- est dose of toxin (P<0.0001) where the cell viability was also reduced (Fig. 4).
HT-2 toxin caused no significant effect on hormone produc- tion at lower levels. Progesterone and estradiol concentrations decreased significantly (P<0.05) after the exposure to 5 ng/ml and a significant reduction in testosterone, and cortisol levels (P<0.0001) was observed with 50 ng/ml toxin concentration, corresponding to reduced cell viability at this toxin concentration (Fig. 5).
3.3. Nuclear receptor RGA agonism and antagonism
No agonistic response was observed for DON, T-2 and HT-2 with any of the reporter gene assay cell lines, at any of the con- centrations tested. An antagonistic effect was seen for DON, T-2 and HT-2 with all the receptors at the higher concentrations tested (Supplementary data, Fig. S.1-3). However, when these results were evaluated together with the cytotoxicity results (Fig. 2), any reduc- tion in binding of the steroid hormones to their relevant receptors appeared to be caused by a reduction in cell viability rather than by interactions with the steroid hormone receptors.
3.4. Gene expression
Thirteen of the 16 steroidogenic genes analyzed in this study were significantly regulated (P<0.05) by the trichothecenes (Fig. 6). The chosen doses for gene expression analysis (100 ng/ml DON, 0.5 ng/ml T-2 toxin, 5 ng/ml HT-2 toxin) had effects in hormone
(a) Mean Progesterone concentration
(b) Mean Estradiol concentration
3,0
Mean P4 conc (ng/ml)
2,5
Mean Estradiol conc (pg/ml)
a
a
2,0
b
2,5
a
a
2,0
1,5
a
a
a
a
a
1,5
1,0
1,0
0,5
b
0,5
b
0,0
0
DON concentration (ng/ml)
0,1
100
0,0
1
10
1000
0
0,1
1
10
100
1000
DON concentration (ng/ml)
(c) Mean Testosterone concentration
(d) Mean Cortisol concentration
5,0
Mean Cortisol conc (ng/ml)
60
a
Mean T conc (ng/ml)
50
a
a
a
a
a
4,0
a
a
a
b
40
3,0
30
2,0
c
b
20
1,0
10
0,0
0
0
0,1
1
10
100
1000
0
0,1
1
10
100
1000
DON concentration (ng/ml)
DON concentration (ng/ml)
1,5
(a) Mean Progesterone concentration
Mean P4 conc (ng/ml)
b
Mean Estradiol conc (pg/ml)
100
(b) Mean Estradiol concentration
a
80
a
a
a
1,2
a
a
a
a
a
a
0,9
60
0,6
40
b
0,3
20
0,0
0
0
0,0005
0,005
0,05
0,5
5
0
0,0005
0,005
0,05
0,5
5
T-2 Toxin concentration (ng/ml)
T-2 Toxin concentration (ng/ml)
60
(c) Mean Testosterone concentration
60
(d) Mean Cortisol concentration
a
a
50
50
a
a
a
Mean T conc (ng/ml)
Mean Cortisol conc (ng/ml)
a
40
40
30
ab
ab
30
b
20
b
b
20
10
b
10
0
0
0,0005
0,005
0,05
0,5
5
0
0
0,0005
0,005
0,05
0,5
5
T-2 Toxin concentration (ng/ml)
T-2 Toxin concentration (ng/ml)
production and no significant effects in cell viability. Whereas HMGR and CYP19 were down-regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. Apart from the above mentioned commonly regulated genes, exposure to DON further resulted in an up-regulation of expression of CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulation of NR5A1; T-2 toxin
caused down-regulation of expression of NR0B1 and NR5A1, while HT-2 toxin resulted in up-regulation of expression of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not sig- nificantly affected by any of the trichothecenes in the present study.
(a) Mean Progesterone concentration
(b) Mean Estradiol concentration
2,0
a
a
a
a
a
a
b
Mean P4 conc (ng/ml)
Mean Estradiol conc (pg/ml)
300
a
a
a
1,6
250
b
200
1,2
150
0,8
100
c
0,4
50
0,0
0
0
0,005
0,05
0,5
5
50
0
0,005
0,05
0,5
5
50
HT-2 Toxin concentration (ng/ml)
HT-2 Toxin concentration (ng/ml)
(c) Mean Testosterone concentration
(d) Mean Cortisol concentration
2,1
a
a
a
70
a
a
60
a
a
Mean T conc (ng/ml)
1,8
Mean Cortisol conc (ng/ml)
ab
ab
1,5
50
b
1,2
b
40
0,9
30
C
0,6
20
0,3
10
0,0
0
0
0,005
0,05
0,5
5
50
0
0,005
0,05
0,5
5
50
HT-2 Toxin concentration (ng/ml)
HT-2 Toxin concentration (ng/ml)
DON 100 ng/ml
T-2 toxin 0.5 ng/ml
HT-2 toxin 5 ng/ml
1.5
Log2(fold change) compared with control, 0.1% MeOH
1.0
0.5
0.0
H
”
-0.5
-1.0
CYP1A1
HMGR
EPHX-
MC2R-
NR5A1
NROB1.
StAR
CYP11A-
HSD3B2
CYP17
CYP21
HSD17B1.
HSD17B4-
CYP11B1
CYP11B2
CYP19.
4. Discussion
In the present study, the trichothecenes DON, T-2 and HT-2 toxins have been evaluated for their potential to cause endocrine disruption by altering gene expression, hormone production and interfering with nuclear receptor signalling.
The T-2 concentrations used in the different assays in this manuscript are in the same range as those expected in vivo in the 1st to 2nd h after application according to kinetic modelling, which we have performed on the basis of total 3H-T2-blood con- centrations measured after oral application of 0.5 mg/kg to chickens (Chi et al., 1978). Extending the model to H-T2, a major metabolite of T2, we assume that comparable conditions and concentration ratios are valid in the assays used in the present study. DON serum levels have been directly measured in experiments with pigs. Feed- ing of approximately 20 mg of DON resulted in serum levels of 10-20 ng/ml (Goyarts and Dänicke, 2006), which is comparable to the lower doses tested in this study.
4.1. Cell viability and cytotoxicity
Cytotoxic effects of trichothecenes have been observed in both in vitro and in vivo studies. In eukaryote cells trichothecenes are responsible for multiple inhibitory effects. They may inhibit pro- tein, DNA and RNA synthesis, inhibit mitochondrial function, have effects on cell division and alter the cell membrane and induce apo- ptosis (Rocha et al., 2005). Protein synthesis inhibition is a primary toxic effect of trichothecenes (Mclaughlin et al., 1977) and as cell metabolism depends on this, many of the other observed effects may be a consequence of this.
Four colorimetric bioassays have been utilized in the present study including AlamarBlue, NR, MTT and BrdU assay to study the viability/cytotoxicity of trichothecenes. Whereas MTT and Alamar- Blue are specific tests for mitochondrial metabolic activity of viable cells (Mosmann, 1983), NR quantifies membrane integrity using the ability of viable cells to accumulate the dye in the lysosomes, and exploiting that the measure of dye extracted from the cells is linear with cell numbers (Borenfreund and Puerner, 1985). The BrdU bioassay on the other hand quantifies the amount of DNA
synthesized by the dividing cells and can be a good measure of cell proliferation. The present study reports no significant differ- ence between BrdU, NR and AlamarBlue assays at concentrations tested on H295R cells. Some studies have indicated some superi- ority of the BrdU assay as compared to the MTT and LDH bioassays for the analysis of trichothecenes in 3T3 cells (Widestrand et al., 1999), whereas NR uptake was superior to MTT and AlamarBlue in porcine pulmonary alveolar macrophages exposed to DON (Döll et al., 2009).
In the H295R cells effects of the trichothecenes were investi- gated by AlamarBlue, NR and BrdU assays, where a dose related decrease in cell viability was observed. The highest toxin concen- trations used (1000 ng/ml DON, 5 ng/ml T-2 toxin and 50 ng/ml HT-2 toxin), caused significant reduction of viable cells to less than 70% compared to the unexposed cells. Inhibition of DNA synthe- sis has been demonstrated by the dose response curves obtained using the BrdU bioassay especially in the highest concentrations of trichothecenes tested. Studies have shown that DON in high doses (>100 ng/ml, equivalent to 0.34 µM), inhibits DNA synthe- sis (Ranzenigo et al., 2008), which corresponds to our results and may explain the reduced cell viability in the present study. T-2 toxin has been reported to cause a dose-related inhibition of growth of ovarian granulosa cells in rats by inducing apoptosis and increasing reactive oxygen species (Wu et al., 2011).
The MTT bioassay was used to assess cell metabolism as a mea- sure of viability in the RGA cell lines following exposure to DON, T-2 and HT-2 toxins. The highest concentrations of the mycotoxins tested were capable of impairing cell viability. Regarding cytotoxic strength, an order of potency of T-2> HT-2>DON was observed. This order of toxicity is similar to that of previous observations (Gutleb et al., 2002). Two mammalian cell lines (K-562 and MIN- GL1) have shown T-2 to be the most cytotoxic in comparison to other Fusarium mycotoxins by the MTT test with a 50% inhibitory concentration (IC50) of less than 1 ng/ml (Visconti et al., 1991).
4.2. Hormone production
H295R cells have been used in the present study to evaluate the effects of trichothecenes in steroidogenesis. Forskolin has
been previously established as a stimulant for this cell model (Hilscherova et al., 2004; Zimmer et al., 2011) and in the present study, exposure to 10 p.M Forskolin resulted in increased hormone levels as expected. In the present study, we observed a general dose dependent reduction in hormone levels in media of exposed cells. The reduced cell viability with increasing toxin concentrations partly explains the reduced hormone levels in all studied tri- chothecenes at their highest dose used. Previously, Ranzenigo et al. (2008) observed that DON caused reduction in estradiol production at high doses of the toxin due to its effect on aromatase activity.
Progesterone hormone patterns following exposure to DON and T-2 toxin were biphasic with lower doses reducing hormone levels and higher doses showing increased hormone levels. Cells exposed to 1000 ng/ml DON had progesterone concentration increased sim- ilar to recently reported findings (Medvedova et al., 2011) that DON increased the progesterone release in porcine granulosa cells at 1000 ng/ml but not in the lower doses. The observed increase in progesterone concentration following exposure to 5 ng/ml T-2 toxin differ from a previous report (Caloni et al., 2009), where the toxin inhibited progesterone release by porcine granulosa cells in a dose dependent manner. The authors observed a 30% inhibition of progesterone production after exposure to 0.3 ng/ml T-2 toxin, whereas 30 and 300 ng/ml completely inhibited the FSH plus IGF- I induced progesterone production. On the other hand, T-2 toxin caused a dose dependent inhibition in testosterone production, as reported recently in male rabbits (Kovacs et al., 2011).
4.3. Reporter gene assay
This study found that there is no (ant)agonist activity of these trichothecenes with the androgen, estrogen, progestagen or gluco- corticoid steroid hormone receptors. Despite an apparent reduction in the binding of the steroid hormones to their relevant receptors at the highest concentrations in the antagonistic test, the cytotoxicity data would suggest that this is due to a reduction in cell viability rather than any interactions with the steroid hormone receptors.
4.4. Gene expression vs steroidogenesis
Steroidogenesis is a complex process involving many enzymes and can be interfered at any level leading to changes in the rate of production and concentration of hormones (Hilscherova et al., 2004). H295R cells have been used to evaluate the ability of some endocrine disrupting compounds to alter gene expres- sion (Hilscherova et al., 2004; Zhang et al., 2005; Gracia et al., 2006). Quantitative Real Time RT-PCR was used to determine the potential of the trichothecenes to modulate the expression of important genes in steroidogenesis in H295R cells. Our group (Zimmer et al., 2011) has previously included 5 p.M Forskolin as a positive control to evaluate gene expression in H295R cells where it induced up-regulation of HMGR, ACTHR, StAR, CYP11A, HSD3B2, CYP17, CYP21 and CYP11B2 similar to earlier reports (Hilscherova et al., 2004; Gracia et al., 2006). DON, T-2 and HT-2 toxins at 100 ng/ml (3.38 µM), 0.5 ng/ml (1.07 nM) and 5 ng/ml (11.79 nM) respectively were used for gene expression analysis. At these indi- vidual doses, there were no significant effects on cell viability. The trichothecenes induced the expression of most of the steroidogenic genes analyzed in this study indicating that gene expression alter- ations partly explain the observed patterns in steroidogenesis.
HMGR, hydroxyl-methyl-glutaryl CoA reductase is an enzyme catalyzing the conversion of HMG CoA to mevalonic acid, a rate- limiting step in the control of cholesterol biosynthesis. Cholesterol is extremely important as it is the precursor for the synthesis of all the steroid hormones. In the body, both dietary and de-novo synthesized cholesterol are transported as lipoprotein particles in circulation. The expression of HMGR gene was down-regulated by
all three trichothecenes and thus could result in reduced available cholesterol and partly explain the overall reduction in the steroid hormone synthesis.
CYP1A1 encodes for cytochrome P450 proteins involved in NADPH-dependent electron transport pathway and catalyzes a variety of reactions including cholesterol and steroids synthesis. The expression of CYP1A1 was up-regulated in cells exposed to all the trichothecenes. Recently, T-2 toxin has been reported to induce CYP1A1 in the human intestinal epithelial cell line Caco-2 (Kruber et al., 2011) but caused no effect on CYP1A1 expression in porcine primary hepatocytes (Wang et al., 2011).
MC2R (also called ACTHR) codes for an adrenocorticotropic hormone receptor (Sewer et al., 2007) and is thus important in steroidogenesis. This gene was not significantly regulated by any of the trichothecenes. The H295R cells in the present study were, however, not stimulated by ACTH. Therefore, the reduced hormone induction may not be explained by the regulation of MC2R gene. H295R cells do not respond to ACTH probably due to low expres- sion of the MC2R receptor under normal culture conditions (Rainey et al., 1993).
StAR, steroidogenic acute regulatory protein plays a key role in steroidogenesis as it mediates the transfer of cholesterol to the inner mitochondrial membrane, where it is cleaved to pregne- nolone by the action of CYP11A (cholesterol side chain cleavage) (Payne and Hales, 2004). Although the expression of mRNA for StAR protein was reduced by exposure to DON and HT-2 toxin, this was not statistically significant and may not necessarily explain the reduced hormone induction.
CYP11A is located in the inner mitochondrial membrane and catalyzes the conversion of cholesterol to pregnenolone, the first rate-limiting step in steroid hormone synthesis (Payne and Hales, 2004). The expression of CYP11A gene was significantly down- regulated by HT-2 toxin and partially by DON and T-2 toxin exposed cells. The reduced expression of this important gene might partly explain the overall reduction in hormone production. A similar observation was reported in DON exposed cells (Ranzenigo et al., 2008), where the toxin inhibited FSH plus IGF-I-induced CYP11A1 mRNA abundance in porcine granulosa cells.
HSD3B2 is a bifunctional enzyme catalyzing the oxidative conversion of delta(s)-ene-3-beta hydroxysteroid and oxidative conversion of ketosteroids. Enzymatic activities of 3ßHSD are essential for the production of all active steroid hormones i.e. progesterone from pregnenolone, 17a-hydroxyprogesterone from 17a-hydroxypregnenolone and androstenedione from dehy- droepiandrostenedione (Payne and Hales, 2004). In the present study the expression of HSD3B2 was up-regulated in DON exposed cells, although this up-regulation did not translate to increased progesterone production under the used conditions.
CYP21 catalyses the 21-hydroxylation of steroids and is required for the adrenal synthesis of mineralocorticoids and glucocorti- coids. It is responsible for the conversion of progesterone and 17a-hydroxyprogesterone to deoxycorticosterone and deoxycor- tisol respectively. In this study, the three trichothecenes induced up-regulation of the expression of CYP21 but the regulation did not reflect its activity in the production of cortisol hormone.
CYP19 (aromatase) is an enzyme responsible for catalyzing the last steps of estrogen biosynthesis. The down-regulation of CYP19 expression by the three trichothecenes partly could explain the reduced levels of estrogen hormone. DON has been reported to inhibit FSH plus IGF-I-induced CYP19A1 expression in porcine granulosa cells (Ranzenigo et al., 2008) thus inhibiting estradiol production. While the three trichothecenes inhibited aromatase activity (and therefore decreased production of estradiol from androstenedione and testosterone), HT-2 toxin induced the up- regulation of HSD17B1 (increased activity of conversion of estrone to estradiol and androstenedione to testosterone).
DON induced the up-regulation of CYP17, whereas it was down-regulated by exposure to HT-2 toxin. CYP17 is involved in the conversion of pregnenolone and progesterone to their respective 17a-hydroxylated products and subsequently to dehy- droepiandrostenedione and androstenedione. It catalyzes both the 17a-hydroxylation and the 17,20-lyase reaction (Payne and Hales, 2004). The up-regulation of CYP17, however, did not translate to increased hormone production in the DON-exposed cells.
HT-2 toxin up-regulated the EPHX gene but T-2 toxin did not cause a significant effect similar to recent findings (Wang et al., 2011), where EPHX was up-regulated by HT-2 but not by T-2 toxin exposure in porcine hepatocytes hepG2 cells.
In conclusion, the present findings indicate that the exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, DNA synthesis and steroidogenesis in a dose-dependent man- ner. Although the trichothecenes did not exhibit any endocrine disrupting activity via the androgen, estrogen, progestagen or glu- cocorticoid receptors alteration in gene expression and hormone synthesis indicated their potential to act as endocrine disruptors.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was funded by NFR grant 199412/199 “Mycotoxin contamination in Norwegian food and feed - Modelling, reduc- tive approach and risk assessment with regards to the whole food chain”. The authors wish to express gratitude to Karin Zimmer, Ingrid Olsaker and Camilla Carlson for the design of primers.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.toxlet.2012.09.005.
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