Browsing by Author "Ndossi, D."
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Item Effects of mycotoxins on hormone production in primary Leydig cells isolated from pigs(Lysbilde, 2013) Ndossi, D.; Kalayou, S.; Groseth, P. K.; Ropstad, E.; Verhaegen, S.Mycotoxins are toxins produced by fungi in many agricultural products worldwide either pre- or post-harvesting. Fusarium species are among the most researched plant pathogenic fungi (1) that produce a number of mycotoxins including DON, NIV, T-2, HT-2, fumonisins, ZEN and its metabolites α- and β-zearalenol (2). Some fungi are able to produce more than one mycotoxin, but also multiple fungi can contaminate the same crop (3). Natural co-occurrence of Fusarium mycotoxins increases the concern on the exposure to mixtures of mycotoxins e.g. co-occurrence of DON/ ZEN/ T-2/ HT-2 (4,5). Exposure to mycotoxins can result in a variety of health effects, ranging from acute toxic response to potential long-term carcinogenic and teratogenic effects (6). Effects of mycotoxins on reproduction in livestock have been reviewed extensively (7). ZEN ingestion in animals is associated with anestrus, abortion, increased embryonic and fetal death, increased stillbirths, reduced milk production, hyperestrogenism and poor quality semen (7,8) In vitro, ZEN increased the progesterone production in porcine granulosa cells (9). ZEN and α-ZOL have potent estrogenic effects and promote hormone production in H295R cells (10). DON, T-2 and HT-2 reduced cell viability, inhibit steroidogenesis and alter expression of steroidogenic genes in human adrenocarcinoma (H295R) cells (11) Leydig cells are the testicular endocrine cells capable of producing steroid hormones. Pig Leydig cell culture is a good in vitro model to study steroidogenesis and screen effects of some chemicals (12,13). We hypothesize that F. graminearum culture extracts contain a mixture of naturally co-occurring mycotoxins that are able to cause deleterious effects in vitro in Leydig cells.Item Effects of mycotoxins on hormone production in primary Leydig cells isolated from pigs(Lysbilde, 2013) Ndossi, D.; Kalayou, S.; Groseth, P. K.; Ropstad, E.; Verhaegen, S.Mycotoxins are toxins produced by fungi in many agricultural products worldwide either pre- or post-harvesting. Fusarium species are among the most researched plant pathogenic fungi (1) that produce a number of mycotoxins including DON, NIV, T-2, HT-2, fumonisins, ZEN and its metabolites α- and β-zearalenol (2). Some fungi are able to produce more than one mycotoxin, but also multiple fungi can contaminate the same crop (3). Natural co-occurrence of Fusarium mycotoxins increases the concern on the exposure to mixtures of mycotoxins e.g. co-occurrence of DON/ ZEN/ T-2/ HT-2 (4,5). Exposure to mycotoxins can result in a variety of health effects, ranging from acute toxic response to potential long-term carcinogenic and teratogenic effects (6). Effects of mycotoxins on reproduction in livestock have been reviewed extensively (7). ZEN ingestion in animals is associated with anestrus, abortion, increased embryonic and fetal death, increased stillbirths, reduced milk production, hyperestrogenism and poor quality semen (7,8) In vitro, ZEN increased the progesterone production in porcine granulosa cells (9). ZEN and α-ZOL have potent estrogenic effects and promote hormone production in H295R cells (10). DON, T-2 and HT-2 reduced cell viability, inhibit steroidogenesis and alter expression of steroidogenic genes in human adrenocarcinoma (H295R) cells (11) Leydig cells are the testicular endocrine cells capable of producing steroid hormones. Pig Leydig cell culture is a good in vitro model to study steroidogenesis and screen effects of some chemicals (12,13). We hypothesize that F. graminearum culture extracts contain a mixture of naturally co-occurring mycotoxins that are able to cause deleterious effects in vitro in Leydig cells.Item Efficacy of CIDR treatment against ovarian follicular cysts in cows(Kagoshima University, 2004) Ndossi, D.; Kamimura, S.; Ando, T.; Iwakuma, A.; Hamana, K.; Matsuzaki, K.; Nojiri, Y.; Matsushita, T.; Kaizo, S.The objectives of this study were to identify cows with follicular cysts and study the efficacy of CIDR (Progesterone=1.9g) treatment against ovarian follicular cysts in Japanese Black and Hoistein cows. Forty-one cows from Kagoshima Prefecture and Miyazaki Prefecture were initially identified by rectal palpation to have follicular cysts. These were randomly allocated into three groups where group 1 (n=29) were treated with CIDR for 7 days, group 2 (n=11) treated with CIDR for 14 days, and group 3 (n=1) as the control group where no CIDR was inserted. After removal of CIDR, all animals received an intramuscular injection of 25 mg PGF_<2α>. Fifteen out of 41 cows were confirmed to have follicular cysts after plasma progesterone determination by radioimmunoassay, as they had a pre-treatment progesterone concentration of<1ng/ml. Four Holstein cows were treated with 7 days of insertion. The plasma progesterone concentration increased significantly (P<0.01) after CIDR treatment i. e. day 0 (0.32±0.22ng/ml) to day 21 (3.96±1.99ng/ml). Eight Japanese Black cows with a pre-treatment mean plasma progesterone concentration of 0.32±0.26ng/ml were treated with CIDR for 7 days where 7 of them (87.5%) had an increased post-treatment progesterone concentration of 3.66±1.54ng/ml (P<0.01). Three Japanese Black cows treated with 14 days of CIDR insertion had pre-treatment and post-treatment progesterone concentrations of 0.35±0.14ng/ml and 2.40±1.86ng/ml, respectively (P=0.09). Fourteen out of fifteen ovarian follicular cyst cases had increased progesterone levels 14 days after CIDR removal (post-treatment period), 11 cows (73.3%) came to estrus 2-6 days after removal of the CIDR device and ovulation of a dominant follicle occurred with following formation of the corpus luteum. The overall success rate of CIDR treatment against ovarian follicular cysts (increase in plasma progesterone concentration>1ng/ml) was 93.3% in both treated groups.Item Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis(Elsevier Ireland Ltd., 2011-07-23) Frizzell, C.; Ndossi, D.; Verhaegenb, S.; Dahlb, E.; Eriksend, G.; Sørlie, M.; Ropstadb, E.; Mullerf, M.; Elliott, C.; Connollya, L.The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol ( -ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ∼70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.Item An in vitro investigation of endocrine disrupting effects of the mycotoxin alternariol(Elsevier Ltd., 2013) Frizzell, C.; Ndossi, D.; Kalayou, S.; Eriksen, G. S.; Verhaegen, S.; Sørlie, M.; Elliott, C. T.; Ropstad, E.; Connolly, L.Alternariol (AOH) is a mycotoxin commonly produced by Alternaria alternata on a wide range of foods. Few studies to date have been performed to evaluate the effects of AOH on endocrine activity. The present study makes use of in vitro mammalian cellular based assays and gene expression to investigate the ability of AOH to act as an endocrine disruptor by various modes of action. Reporter gene assays (RGAs), incorporating nat- ural steroid hormone receptors for oestrogens, androgens, progestagens and glucocorticoids were used to identify endocrine disruption at the level of nuclear receptor transcriptional activity, and the H295R steroido- genesis assay was used to assess endocrine disruption at the level of gene expression and steroid hormone production. AOH exhibited a weak oestrogenic response when tested in the oestrogen responsive RGA and binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of AOH. H295R cells when exposed to 0.1–1000 ng/ml AOH, did not cause a significant change in testosterone and cortisol hormones but exposure to 1000 ng/ml (3.87 μM) AOH resulted in a significant in- crease in estradiol and progesterone production. In the gene expression study following exposure to 1000 ng/ml (3.87 μM) AOH, only one gene NR0B1 was down-regulated, whereas expression of mRNA for CYP1A1, MC2R, HSD3B2, CYP17, CYP21, CYP11B2 and CYP19 was up-regulated. Expression of the other genes investigated did not change significantly. In conclusion AOH is a weak oestrogenic mycotoxin that also has the ability to interfere with the steroidogenesis pathway.Item An investigation of the endocrine disrupting potential of enniatin B using in vitro bioassays(Elsevier Ltd., 2015) Kalayou, S.; Ndossi, D.; Frizzell, C.; Groseth, P. K.; Connolly, L.; Sørlie, M.; Verhaegen, S.; Ropstad, E.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 (0.01–100 m M) was cytotoxic in the H295R and LC models used; following 48 h incubation with 100 m M. Flow cytometry analysis showed that ENN B exposure (0.1– 25 m M) led to an increased proportion of cells in the S phase at higher ENN B doses (>10 m M) while cells at G 0 /G 1 phase were reduced. At the receptor level, ENN B (0.00156–15.6 m M) did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs), however cell viability was affected at 15.6 m M. 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 (100 m M) 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 (10 m M) compared to the control. Genes HMGR, StAR, CYP11A, 3 b HSD2 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 (10 m M) in the H295R cells suggests that adrenal endocrine toxicity is an important potential hazard.Item An investigation of the endocrine disrupting potential of enniatin B using in vitro bioassays(ELSEVIER, 2015-01) Kalayou, S.; Ndossi, D.; Frizzell, C.; Groseth, P.; Connolly, L.; Sørlie, M.; Verhaegen, S.; Ropstad, E.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 (0.01–100 m M) was cytotoxic in the H295R and LC models used; following 48 h incubation with 100 m M. Flow cytometry analysis showed that ENN B exposure (0.1– 25 m M) led to an increased proportion of cells in the S phase at higher ENN B doses (>10 m M) while cells at G 0 /G 1 phase were reduced. At the receptor level, ENN B (0.00156–15.6 m M) did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs), however cell viability was affected at 15.6 m M. 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 (100 m M) 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 (10 m M) compared to the control. Genes HMGR, StAR, CYP11A, 3 b HSD2 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 (10 m M) in the H295R cells suggests that adrenal endocrine toxicity is an important potential hazard.Item Predicting body weight of Tanzania shorthorn zebu cattle using heart girth measurements(Livestock Research for Rural Development, 2011-04-01) Kashoma, I.; Luziga, C.; Werema, C.; Shirima, G.; Ndossi, D.Impact of research findings on Tanzania shorthorn zebu (TSHZ) cattle under agropastoral system in Tanzania have been lessened due to difficulties in relating visual appearance of animals with their live body weights. Managerial decisions have therefore been based on rough and inaccurate estimates. A simple, workable and accurate means was therefore thought to predict live weight of zebu cattle using heart girth measurement. A total of 300 TSHZ cattle were subjected to weighbridge as well as heart girth measurements and the findings subjected to regression analysis. Their live weights ranged from 170 to 390 Kg. The study revealed that heart girth had high correlation coefficient with live weight (r = 0.94, p<0.01). Grouping of data according to sex indicated that heart girth and live weight had closest correlation coefficient in both male and female, r = 0.9385 and r = 0.9318, respectively. The regression analysis of live weight on heart girth was highly significant (R² = 0.88, P<0.01), and the regression lines indicated that a linear relationship existed. The general equation for prediction of live weight of the TSHZ cattle was as follows: Y = 4.55 X – 409 (± 17.9), Where, Y = live weight (Kg), X = heart girth (cm). This implies that the use of the heart girth measurements in prediction of live weight of TSHZ cattle is credible.Item Predicting body weight of Tanzania shorthorn zebu cattle using heart girth measurements(Livestock Research for Rural Development, 2011-04-01) Kashoma, I. P. B.; Luziga, C.; Werema, C. W.; Shirima, G. A.; Ndossi, D.Impact of research findings on Tanzania shorthorn zebu (TSHZ) cattle under agro- pastoral system in Tanzania have been lessened due to difficulties in relating visual appearance of animals with their live body weights. Managerial decisions have therefore been based on rough and inaccurate estimates. A simple, workable and accurate means was therefore thought to predict live weight of zebu cattle using heart girth measurement. A total of 300 TSHZ cattle were subjected to weighbridge as well as heart girth measurements and the findings subjected to regression analysis. Their live weights ranged from 170 to 390 Kg. The study revealed that heart girth had high correlation coefficient with live weight (r = 0.94, p<0.01). Grouping of data according to sex indicated that heart girth and live weight had closest correlation coefficient in both male and female, r = 0.9385 and r = 0.9318, respectively. The regression analysis of live weight on heart girth was highly significant (R² = 0.88, P<0.01), and the regression lines indicated that a linear relationship existed. The general equation for prediction of live weight of the TSHZ cattle was as follows: Y = 4.55 X – 409 (± 17.9), Where, Y = live weight (Kg), X = heart girth (cm). This implies that the use of the heart girth measurements in prediction of live weight of TSHZ cattle is credible.Item Relative quantification of the proteomic changes associated with the mycotoxin zearalenone in the H295R steroidogenesis model(Elsevier Ltd, 2011) Busk, Ø. L.; Ndossi, D.; Verhaegen, S.; Connolly, L.; Eriksen, G.; Ropstad, E.; Sørlie, M.Zearalenone (ZEN) is a mycotoxin with endocrine disrupting effects having vast economic implications in e.g. pig farming. Structurally, ZEN resembles 17 b -estradiol, and thus is able to bind to estrogen receptors (ER) in target cells. Because of this, it is also classified as a non-steroidal estrogen, a phytoestrogen, a mycoestrogen, and a growth promoter. Quantitative proteomic analysis was undertaken using stable-isotope labeling by amino acids in cell culture (SILAC) upon exposure of the steroidogenesis cell model H295R with ZEN to elucidate its effect on protein regulation. ZEN significantly regulated 21 proteins, including proteins with known endocrine disrupting effects and several oncogenes. In addition, network analysis using Ingenuity Pathway Analysis showed that ZEN affected the oxidative phosphorylation pathway and the mitochondrial dysfunction pathway, both previously reported to be involved in endocrine dysfunction.Item Using SILAC proteomics to investigate the effect of the mycotoxin, alternariol, in the human H295R steroidogenesis model(Springer Science+Business Media Dordrecht 2014, 2014-11) Kalayou, S.; Hamre, A. G.; Ndossi, D.; Connolly, L.; Sørlie, M.; Ropstad, E.; Verhaegen, S.The mycotoxin alternariol (AOH) is an impor- tant contaminant of fruits and cereal products. The current study sought to address the effect of a non-toxic AOH concentration on the proteome of the steroidogenic H295R cell model. Quantitative proteomics based on stable iso- tope labeling by amino acids in cell culture (SILAC) coupled to 1D-SDS-PAGE-LC-MS/MS was applied to subcellular-enriched protein samples. Gene ontology (GO) and ingenuity pathway analysis (IPA) were further carried out for functional annotation and identification of protein interaction networks. Furthermore, the effect of AOH on apoptosis and cell cycle distribution was also determined by the use of flow cytometry analysis. This work identified 22 proteins that were regulated significant- ly. The regulated proteins are those involved in early stages of steroid biosynthesis (SOAT1, NPC1, and ACBD5) and C21-steroid hormone metabolism (CYP21A2 and HSD3B1). In addition, several proteins known to play a role in cellular assembly, organization, protein synthesis, and cell cycle were regulated. These findings provide a new framework for studying the mechanisms by which AOH modulates steroidogenesis in H295R cell model.