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The in vitro effects of Fusarium mycotoxins on bovine granulosa cell CYP11A1 and CYP19A1 mRNA
Abstracts / Toxicology Letters 229S (2014) S40–S252
Integration of multiple genotoxicity endpoints into repeat-dose rodent studies provides broad assessment of genotoxic potential of different modes of action while reducing the use of animals. Using male Lambda LIZ/lacI C57BL/6 homozygous (Big Blue® ) transgenic mice, we examined the induction of cII mutations in liver and bone marrow, micronuclei in blood reticulocytes and mature erythrocytes (mnRET and mnNCE, respectively), and Pig-a mutant phenotype reticulocytes and erythrocytes (RETCD24− and RBCCD24− , respectively). Animals were treated with olive oil (5 mL/kg/day for 28 days), BaP (50 mg/kg/day for 28 days), or ENU (40 mg/kg/day on Days 1, 2 and 3), with sacrifice on Day 31. Significant increases (p < 0.001) in cII mutant frequencies were observed for BaP in liver (4.89-fold) and bone marrow (4.15-fold), and for ENU in liver (17.7fold) and bone marrow (10.6-fold). Peripheral blood was collected ∼3 h before sacrifice and analyzed for Pig-a mutant phenotype and micronucleus frequencies by flow cytometry using MutaFlow® and MicroFlow® kits, respectively (Litron Laboratories). BaP induced significant increases (p < 0.001) in mnRET, mnNCE, RETCD24− and RBCCD24− (1.56-, 2.11-, 616-, and 101-fold, respectively). ENU induced significant increases (p < 0.001) in mnNCE, RETCD24− and RBCCD24− (1.40-, 1050-, and 208-fold, respectively). As expected, mnRET were not elevated by ENU (p > 0.05) due to the long interval between last administration and blood harvest. This study demonstrates the robustness of the Big Blue® Mouse Mutation Assay to detect induced mutagenesis by a direct acting mutagen and one requiring metabolic activation. This work also demonstrated the ability to integrate additional endpoints for mutation and clastogenicity. http://dx.doi.org/10.1016/j.toxlet.2014.06.231 P-1.52 The in vitro effects of Fusarium mycotoxins on bovine granulosa cell CYP11A1 and CYP19A1 mRNA Fabiola Pizzo 1 , Francesca Caloni 1,∗ , Nicole Schreiber 2 , Cristina Cortinovis 1 , Morgan Totty 2 , Leon J. Spicer 2 1
Università degli Studi di Milano, Department of Health, Animal Science and Food Safety (VESPA), Milan, Italy, 2 Oklahoma State University, Department of Animal Science, Stillwater, USA Zearalenone (ZEA), a phyto-estrogenic molecule, and Deoxynivalenol (DON) are mycotoxins produced by Fusarium spp. and found to co-exist in grain commodities. Reproductive effects caused by ZEA in ruminants have been reported, while direct effects on fertility related to DON are unknown. The aim of this study is to evaluate the effects of ZEA hydroxylated metabolites, ␣-Zearalenol (␣-ZEA) and -Zearalenol (-ZEA), and DON on bovine small granulosa cell (SMGC) CYP11A1 and CYP19A1 mRNA, genes that encode key enzymes of steroid hormone biosynthesis. SMGC were cultured for 2 days in 10% fetal bovine serum-containing medium followed by 2 days in serum-free medium containing control or mycotoxin treatments. Cells were exposed to ␣-ZEA at 3.1 M, ZEA at 31 M and DON at 3.3 M. The effects of ␣-ZEA and -ZEA, at 3.1 M and 31 M respectively, were also tested in presence of DON at 3.3 M. At the end of the exposure period, gene expression of CYP11A1 and CYP19A1 were determined using the quantitative Real Time Polymerase Chain Reaction (RT-PCR) technique. DON (3.3 M), alone and combined with ␣-ZEA and -ZEA, strongly stimulated (P < 0.001) granulosa cell CYP11A1 mRNA abundance whereas ␣-ZEA and -ZEA alone had no effect (P > 0.05) compared to the control. -ZEA at 31 M dramatically (P < 0.001) increased granulosa cell CYP19A1 mRNA abundance while all the other treat-
S57
ments had no effect. Further studies are needed for understanding the effects of Fusarium mycotoxins on granulosa cells. http://dx.doi.org/10.1016/j.toxlet.2014.06.232 P-1.53 Differential effects of Fumonisin B1 and zearalenone metabolites on estradiol and progesterone production in cultured bovine granulosa cells Francesca Caloni 1,∗ , Marco Albonico 1 , Luis Schutz 2 , Fabiola Pizzo 1 , Leon J. Spicer 2 1 Università degli Studi di Milano, Department of Health, Animal Science and Food Safety, Milan, Italy, 2 Oklahoma State University, Department of Animal Science, Stillwater, USA
Fumonisin B1 (FB1) is a mycotoxin frequently occurring in corn in combination with zearalenone (ZEA), an oestrogenic fusariotoxin. The purpose of this study is to compare the effects of ZEA main metabolites, ␣-ZEA and -ZEA, to that of FB1 on ovarian steroidogenesis by using a well-established in vitro bovine granulosa cell (GC) model. FB1, ␣-ZEA and -ZEA were each tested at 1 g/ml. Co-exposures to ␣-ZEA and FB1 or -ZEA and FB1 were also carried out. All experiments (n = 3) were performed with and without IGF1 (30 ng/ml). Concentrations of progesterone (P4) and estradiol (E2) in cell culture medium were determined using radioimmunoassays. A factorial ANOVA revealed that IGF1 increased FSH-induced E2 and P4 production by 4.1- and 2.7fold, respectively. FB1 had no effect (P > 0.20) on FSH- or FSH plus IGF1-induced E2 production. In contrast, both ␣-ZEA and -ZEA increased (P < 0.05) FSH-induced E2 production by 2.6- to 3.2-fold, and inhibited (P < 0.05) FSH plus IGF1-induced E2 production by 38–44%. Averaged across all treatments, P4 production was stimulated by 44% after exposure to FB1. ␣-ZEA and -ZEA had no significant effect on P4 production in the absence or presence of IGF1. In conclusion, the present study indicates that FB1 and ZEA metabolites directly alter ovarian steroid production in cattle, and provides information on the differential effects of FB1 versus ␣-ZEA and -ZEA on bovine granulosa cell steroidogenesis. Studies are in progress to better understand the mechanism of action of FB1, ZEA metabolites on granulosa cell steroidogenesis. http://dx.doi.org/10.1016/j.toxlet.2014.06.233 P-1.54 Mitochondrial thioredoxin system as a primary target for mercury compounds Vasco Branco 1 , Ana Godinho-Santos 1 , João Gonc¸alves 1 , Jun Lu 2 , Arne Holmgren 2 , Cristina Carvalho 1,∗ 1 Instituto de Investigac¸ão do Medicamento (iMed.ULisboa), Faculdade de Farmácia da Universidade de Lisboa, Lisboa, Portugal, 2 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
The thioredoxin system is essential for the maintenance of key cellular functions. The activity of the cytosolic enzymes thioredoxin1 (Trx1) and thioredoxin reductase1 (TrxR1) is known to be inhibited by mercury, but the extent of interaction with mitochondrial isoforms TrxR2 and Trx2 is unknown. This study aimed at understanding how mercury compounds affected the activity of mitochondrial and cytosolic thioredoxin systems and the transcription and expression of TrxR1 and TrxR2. Additionally, the effects of
Integration of multiple genotoxicity endpoints into repeat-dose rodent studies provides broad assessment of genotoxic potential of different modes of action while reducing the use of animals. Using male Lambda LIZ/lacI C57BL/6 homozygous (Big Blue® ) transgenic mice, we examined the induction of cII mutations in liver and bone marrow, micronuclei in blood reticulocytes and mature erythrocytes (mnRET and mnNCE, respectively), and Pig-a mutant phenotype reticulocytes and erythrocytes (RETCD24− and RBCCD24− , respectively). Animals were treated with olive oil (5 mL/kg/day for 28 days), BaP (50 mg/kg/day for 28 days), or ENU (40 mg/kg/day on Days 1, 2 and 3), with sacrifice on Day 31. Significant increases (p < 0.001) in cII mutant frequencies were observed for BaP in liver (4.89-fold) and bone marrow (4.15-fold), and for ENU in liver (17.7fold) and bone marrow (10.6-fold). Peripheral blood was collected ∼3 h before sacrifice and analyzed for Pig-a mutant phenotype and micronucleus frequencies by flow cytometry using MutaFlow® and MicroFlow® kits, respectively (Litron Laboratories). BaP induced significant increases (p < 0.001) in mnRET, mnNCE, RETCD24− and RBCCD24− (1.56-, 2.11-, 616-, and 101-fold, respectively). ENU induced significant increases (p < 0.001) in mnNCE, RETCD24− and RBCCD24− (1.40-, 1050-, and 208-fold, respectively). As expected, mnRET were not elevated by ENU (p > 0.05) due to the long interval between last administration and blood harvest. This study demonstrates the robustness of the Big Blue® Mouse Mutation Assay to detect induced mutagenesis by a direct acting mutagen and one requiring metabolic activation. This work also demonstrated the ability to integrate additional endpoints for mutation and clastogenicity. http://dx.doi.org/10.1016/j.toxlet.2014.06.231 P-1.52 The in vitro effects of Fusarium mycotoxins on bovine granulosa cell CYP11A1 and CYP19A1 mRNA Fabiola Pizzo 1 , Francesca Caloni 1,∗ , Nicole Schreiber 2 , Cristina Cortinovis 1 , Morgan Totty 2 , Leon J. Spicer 2 1
Università degli Studi di Milano, Department of Health, Animal Science and Food Safety (VESPA), Milan, Italy, 2 Oklahoma State University, Department of Animal Science, Stillwater, USA Zearalenone (ZEA), a phyto-estrogenic molecule, and Deoxynivalenol (DON) are mycotoxins produced by Fusarium spp. and found to co-exist in grain commodities. Reproductive effects caused by ZEA in ruminants have been reported, while direct effects on fertility related to DON are unknown. The aim of this study is to evaluate the effects of ZEA hydroxylated metabolites, ␣-Zearalenol (␣-ZEA) and -Zearalenol (-ZEA), and DON on bovine small granulosa cell (SMGC) CYP11A1 and CYP19A1 mRNA, genes that encode key enzymes of steroid hormone biosynthesis. SMGC were cultured for 2 days in 10% fetal bovine serum-containing medium followed by 2 days in serum-free medium containing control or mycotoxin treatments. Cells were exposed to ␣-ZEA at 3.1 M, ZEA at 31 M and DON at 3.3 M. The effects of ␣-ZEA and -ZEA, at 3.1 M and 31 M respectively, were also tested in presence of DON at 3.3 M. At the end of the exposure period, gene expression of CYP11A1 and CYP19A1 were determined using the quantitative Real Time Polymerase Chain Reaction (RT-PCR) technique. DON (3.3 M), alone and combined with ␣-ZEA and -ZEA, strongly stimulated (P < 0.001) granulosa cell CYP11A1 mRNA abundance whereas ␣-ZEA and -ZEA alone had no effect (P > 0.05) compared to the control. -ZEA at 31 M dramatically (P < 0.001) increased granulosa cell CYP19A1 mRNA abundance while all the other treat-
S57
ments had no effect. Further studies are needed for understanding the effects of Fusarium mycotoxins on granulosa cells. http://dx.doi.org/10.1016/j.toxlet.2014.06.232 P-1.53 Differential effects of Fumonisin B1 and zearalenone metabolites on estradiol and progesterone production in cultured bovine granulosa cells Francesca Caloni 1,∗ , Marco Albonico 1 , Luis Schutz 2 , Fabiola Pizzo 1 , Leon J. Spicer 2 1 Università degli Studi di Milano, Department of Health, Animal Science and Food Safety, Milan, Italy, 2 Oklahoma State University, Department of Animal Science, Stillwater, USA
Fumonisin B1 (FB1) is a mycotoxin frequently occurring in corn in combination with zearalenone (ZEA), an oestrogenic fusariotoxin. The purpose of this study is to compare the effects of ZEA main metabolites, ␣-ZEA and -ZEA, to that of FB1 on ovarian steroidogenesis by using a well-established in vitro bovine granulosa cell (GC) model. FB1, ␣-ZEA and -ZEA were each tested at 1 g/ml. Co-exposures to ␣-ZEA and FB1 or -ZEA and FB1 were also carried out. All experiments (n = 3) were performed with and without IGF1 (30 ng/ml). Concentrations of progesterone (P4) and estradiol (E2) in cell culture medium were determined using radioimmunoassays. A factorial ANOVA revealed that IGF1 increased FSH-induced E2 and P4 production by 4.1- and 2.7fold, respectively. FB1 had no effect (P > 0.20) on FSH- or FSH plus IGF1-induced E2 production. In contrast, both ␣-ZEA and -ZEA increased (P < 0.05) FSH-induced E2 production by 2.6- to 3.2-fold, and inhibited (P < 0.05) FSH plus IGF1-induced E2 production by 38–44%. Averaged across all treatments, P4 production was stimulated by 44% after exposure to FB1. ␣-ZEA and -ZEA had no significant effect on P4 production in the absence or presence of IGF1. In conclusion, the present study indicates that FB1 and ZEA metabolites directly alter ovarian steroid production in cattle, and provides information on the differential effects of FB1 versus ␣-ZEA and -ZEA on bovine granulosa cell steroidogenesis. Studies are in progress to better understand the mechanism of action of FB1, ZEA metabolites on granulosa cell steroidogenesis. http://dx.doi.org/10.1016/j.toxlet.2014.06.233 P-1.54 Mitochondrial thioredoxin system as a primary target for mercury compounds Vasco Branco 1 , Ana Godinho-Santos 1 , João Gonc¸alves 1 , Jun Lu 2 , Arne Holmgren 2 , Cristina Carvalho 1,∗ 1 Instituto de Investigac¸ão do Medicamento (iMed.ULisboa), Faculdade de Farmácia da Universidade de Lisboa, Lisboa, Portugal, 2 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
The thioredoxin system is essential for the maintenance of key cellular functions. The activity of the cytosolic enzymes thioredoxin1 (Trx1) and thioredoxin reductase1 (TrxR1) is known to be inhibited by mercury, but the extent of interaction with mitochondrial isoforms TrxR2 and Trx2 is unknown. This study aimed at understanding how mercury compounds affected the activity of mitochondrial and cytosolic thioredoxin systems and the transcription and expression of TrxR1 and TrxR2. Additionally, the effects of