M. Albonico

Evidence for direct effects of glyphosate on ovarian function: glyphosate influences steroidogenesis and proliferation of bovine granulosa but not theca cells in vitro.

Glyphosate (GLY) is a common herbicide used worldwide but its effect on ovarian function in mammals is unknown. The aim of this study was to determine the potential endocrine disruptor effects of GLY on ovarian function evaluating cell proliferation, steroidogenesis and gene expression using bovine granulosa cells (GC) and theca cells as in vitro models. GC proliferation was impaired (P < 0.05) after exposure to GLY at 0.5, 1.7 and 5 μg ml−1. GC progesterone production was not affected (P ≥ 0.05) at all doses tested while estradiol production was inhibited (P < 0.05) by GLY at 5 μg ml−1. At the same concentration GLY showed no effect (P ≥ 0.05) on theca cell proliferation and steroidogenesis. At higher concentrations (0.01 and 0.3 mg ml−1), GLY had no significant effect (P ≥ 0.05) on GC proliferation and steroidogenesis. These studies, for the first time, suggest that GLY may affect the reproductive system in cattle via direct action on ovarian function; however, further studies will be required to understand better the mechanism of action and to determine the in vivo reproductive effects of GLY. Copyright

Direct effects of the algal toxin, domoic acid, on ovarian function: Bovine granulosa and theca cells as an in vitro model

Domoic acid (DA) is a potent neurotoxin produced by alga Pseudo-nitzschia spp. and has been associated with reproductive disorders in mammals. The aim of this study was to investigate if DA can affect the reproductive system via direct action on ovarian function. Bovine granulosa and theca cells were used as in vitro models for evaluating DA effects on ovarian cell proliferation and steroid production. In small-follicle granulosa cells (SMGC), cell proliferation and estradiol (E2) production was not affected (P>0.05) while progesterone (P4) production was inhibited (P<0.05) by DA at all doses tested. In large-follicle granulosa cells (LGGC), DA had no effect (P>0.05) on cell proliferation or P4 production while E2 production was stimulated by 1 and 5 µg/ml DA (P<0.05). DA (1 µg/ml) attenuated (P<0.05) insulin-like growth factor 1 (IGF-1)-induced P4 production by large-follicle theca cells (LGTC), but did not affect androstenedione (A4) production or proliferation of LGTC. In glutamate-free medium, DA inhibited (P<0.05) SMGC E2 production and this inhibition was similar to inhibition of E2 by trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid monohydrate (ACPD; a selective metabotropic glutamate receptor subtype agonist) while kainic acid (KA; an ionotropic glutamate receptor subtype agonist) had no effect (P>0.10) on E2 production. Collectively, these results show for the first time that DA has direct effects on ovarian GC and TC steroidogenesis. Because DA inhibited E2 and P4 production, DA has the potential to be an endocrine disruptor.

In vitro effects of the Fusarium mycotoxins fumonisin B1 and beauvericin on bovine granulosa cell proliferation and steroid production.

ABSTRACT Fusarium mycotoxins are natural contaminants of various commodities representing significant problem worldwide. Since the co‐occurrence of beauvericin (BEA) and fumonisin B1 (FB1) in grains is frequent, the present study was carried out to evaluate the individual and combined effects of FB1 and BEA on cell proliferation, steroid production and gene expression using bovine granulosa cells (GC). When tested alone FB1 did not show (P ≥ 0.05) effects on cell proliferation at any dose. Whereas BEA at 3, 6, and 10 &mgr;M significantly decreased (P < 0.05) cell numbers. FB1 alone had no significant effect (P ≥ 0.05) on progesterone production at any tested doses, whereas FB1 at 1, 1.5 and 3 &mgr;M slightly inhibited (P < 0.05) estradiol production. At concentrations ≥3 &mgr;M, BEA was found to strongly decrease (P < 0.05) steroid production, and FB1 did not influence these effects of BEA. At 10 &mgr;M, both mycotoxins decreased (P < 0.001) serum‐induced GC proliferation. At 30 &mgr;M, BEA showed inhibitory effects on FSH plus IGF1‐induced CYP11A1 and CYP19A1 mRNA abundance (P < 0.05), whereas FB1 at 30 &mgr;M had no effect on CYP11A1 and CYP19A1 gene expression. Taken together these results demonstrate that the Fusarium mycotoxins BEA and FB1 may impair reproductive function in cattle. HIGHLIGHTSGranulosa cells were obtained from bovine ovaries collected at local slaughterhouse.Beauvericin (BEA) drastically decreased bovine granulosa cell number and steroid production.Fumonisin B1 (FB1) slightly inhibited estradiol production.FB1 did not influence the effects of BEA.

In vitro toxicity of beauvericin alone and combined with fumonisin B1 or deoxynivalenol on Caco-2 cells

Beauvericin (BEA) is a mycotoxin produced by Fusarium species, frequently occurring in cereal grains in combination with fumonisin B1 (FB1) and deoxynivalenol (DON). The aim of this study was to evaluate the in vitro toxic effects of BEA alone and combined with FB1 or DON on human intestinal Caco-2 cells cultured on semi-permeable inserts (Caloni et al., 2012). Caco-2 cells were treated for 24 h with BEA (1.5 μM) alone and combined with FB1 (1.5 μM) or DON (3.5 μM) on both apical (Ap) and basolateral (Bl) sides. Barrier impairment was assessed by measuring the trans-epithelial electrical resistance (TEER) after 1 h, 2 h and 24 h of treatment. At the end of the experiment, the culture medium was collected for interleukin-8 (IL-8) determination. The results indicate that TEER was not significantly affected by Ap or Bl exposure to BEA and FB1 alone, whereas a significant decrease (P<0.05) of TEER was observed after exposure to BEA in combination with FB1 for 1 h and 2 h. DON was found to decrease (P<0.05) TEER alone and combined with BEA after Bl application starting from the second hour of treatment. No significant release of the inflammatory mediator IL-8 was observed after Ap or Bl exposure to BEA and FB1 alone and in combination. On the contrary, DON alone and combined with BEA induced a significant (P<0.05) release of IL-8 after both Ap and Bl exposure. Further investigations are underway to better clarify the effects of BEA on the intestinal epithelium and its interaction with other fusariotoxins.

In Vitro toxicological effects of Fumonisin B1 and Beauvericin on bovine granulosa cells

Fumonisin B 1 (FB 1 ) and beauvericin (BEA) are fusariotoxins found to co-exist in food and feed commodities. The aim of this study is to evaluate the individual and combined effects of FB 1 and BEA on bovine granulosa cell proliferation and steroid production. Granulosa cells (GC) from small bovine follicles (1-5 mm) were cultured for 48 hours in 10% fetal bovine serum followed by 48 hours in a serum-free medium containing 500 ng/ml of testosterone (as an estradiol precursor), 30 ng/ml of FSH and 30 ng/ml of IGF-I with and without FB 1 (3 µM) and BEA (3 µM). At the end of the experiment, the numbers of GC were determined using a Coulter counter (Beckman Coulter, USA) and concentrations of progesterone and estradiol in the culture medium were determined by radioimmunoassay. FB 1 and BEA, both individually and in combination, showed an inhibitory effect (P 0.05) on estradiol and progesterone production, whereas BEA (3 µM), both alone and in combination with FB 1 (3 µM), was found to decrease (P < 0.001) the production of both steroids drastically. In conclusion, this in vitro study indicates that FB 1 and BEA, both individually and in combination, may affect GC proliferation to different extents and shows the drastic inhibitory effects of BEA on steroid production.