title: An investigation of the endocrine disrupting potential of enniatin B using in vitro bioassays.
abstract: Evidence that some of the fungal metabolites present in food and feed may act as potential endocrine disruptors is increasing. Enniatin B (ENN B) is among the emerging Fusarium mycotoxins known to contaminate cereals. In this study, the H295R and neonatal porcine Leydig cell (LC) models, and reporter gene assays (RGAs) have been used to investigate the endocrine disrupting activity of ENN B. Aspects of cell viability, cell cycle distribution, hormone production as well as the expression of key steroidogenic genes were assessed using the H295R cell model. Cell viability and hormone production levels were determined in the LC model, while cell viability and steroid hormone nuclear receptor transcriptional activity were measured using the RGAs. ENN B (0.01-100μM) was cytotoxic in the H295R and LC models used; following 48h incubation with 100μM. Flow cytometry analysis showed that ENN B exposure (0.1-25μM) led to an increased proportion of cells in the S phase at higher ENN B doses (>10μM) while cells at G0/G1 phase were reduced. At the receptor level, ENN B (0.00156-15.6μM) did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs), however cell viability was affected at 15.6μM. Measurement of hormone levels in H295R cells revealed that the production of progesterone, testosterone and cortisol in exposed cells were reduced, but the level of estradiol was not significantly affected. There was a general reduction of estradiol and testosterone levels in exposed LC. Only the highest dose (100μM) used had a significant effect, suggesting the observed inhibitory effect is more likely associated with the cytotoxic effect observed at this dose. Gene transcription analysis in H295R cells showed that twelve of the sixteen genes were significantly modulated (p<0.05) by ENN B (10μM) compared to the control. Genes HMGR, StAR, CYP11A, 3βHSD2 and CYP17 were downregulated, whereas the expression of CYP1A1, NR0B1, MC2R, CYP21, CYP11B1, CYP11B2 and CYP19 were upregulated. The reduction of hormones and modulation of genes at the lower dose (10μM) in the H295R cells suggests that adrenal endocrine toxicity is an important potential hazard.
authors.affiliation: Section of Experimental Biomedicine, Norwegian School of Veterinary Science, Oslo, Norway; Mekelle University College of Veterinary Medicine, Mekelle, Ethiopia. Electronic address: shewit.kalayou.teklehaimanot@nmbu.no.
authors.forename: Shewit
authors.initials: S
authors.lastname: Kalayou
authors.affiliation: Sokoine University of Agriculture, Morogoro, Tanzania.
authors.forename: Doreen
authors.initials: D
authors.lastname: Ndossi
authors.affiliation: Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Northern Ireland, UK.
authors.forename: Caroline
authors.initials: C
authors.lastname: Frizzell
authors.affiliation: Section of Experimental Biomedicine, Norwegian School of Veterinary Science, Oslo, Norway.
authors.forename: Per Kristian
authors.initials: PK
authors.lastname: Groseth
authors.affiliation: Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Northern Ireland, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Connolly
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway.
authors.forename: Morten
authors.initials: M
authors.lastname: Sørlie
authors.affiliation: Section of Experimental Biomedicine, Norwegian School of Veterinary Science, Oslo, Norway.
authors.forename: Steven
authors.initials: S
authors.lastname: Verhaegen
authors.affiliation: Section of Experimental Biomedicine, Norwegian School of Veterinary Science, Oslo, Norway.
authors.forename: Erik
authors.initials: E
authors.lastname: Ropstad
mesh_terms: D000818:Animals; D001681:Biological Assay; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D047630:Depsipeptides / Q000633:toxicity*; D052244:Endocrine Disruptors / Q000633:toxicity*; D012739:Gonadal Steroid Hormones / Q000378:metabolism; D006801:Humans; D007985:Leydig Cells / Q000187:drug effects; D008297:Male; D009183:Mycotoxins / Q000633:toxicity*; D009688:Nuclear Reactors; D013256:Steroids / Q000096:biosynthesis; D013552:Swine
chemical_list: D047630:Depsipeptides; D052244:Endocrine Disruptors; D012739:Gonadal Steroid Hormones; D009183:Mycotoxins; D013256:Steroids; C100264:enniatins
keywords: ENN B; Emerging mycotoxins; Endocrine disruption; H295R; Leydig cell; RGA; Steroidogenesis