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Histopathological changes in the gills of freshwater fish, Oreochromis niloticus (L.) Juveniles exposed to Moringa oleifera (Lam.) fresh root-bark extract
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Abstracts / Toxicology Letters 189S (2009) S57–S273
and immune system. Little is understood regarding the specific mechanisms by which PCBs may exert their effects in biological systems. The in vitro BALB/c 3T3 cell transformation assay is a model resembling the in vivo multistep carcinogenesis and has being used for several years to screen the carcinogenicity of chemical and physical agents. We investigated Aroclor 1260 and observed a reduced cloning efficiency of 3T3 cells. The occurrence of morphologically transformed foci was related to the higher dose (20 M). The MTT assay was performed to evaluate the effects of the exposure to a wide range of concentrations (0.001–100 M) of A1260 and two single congeners, PCB 118 and PCB 153, on the proliferative rate of BALB/c 3T3 cells. A1260 induced a toxic effect higher then PCB118 and PCB 153. A global approach utilizing oligo-DNA microarray was used to assess cellular responses to low doses (5, 50 and 500 nM) of A1260 in BALB/c 3T3 cells and to identify genes that are transcriptionally regulated by PCBs exposure. The transcriptional profile makes it possible to analyze real mixtures extracted from environmental matrices and to identify the biomolecular markers directly related to exposure. Acknowledgement: Supported by “Fondazione del Monte di Bologna e Ravenna”, Bologna, Italy. doi:10.1016/j.toxlet.2009.06.586
E18 Pig as a model organism for toxicoproteomic studies on bladder cancer Nisha Verma ∗ , Albert W. Rettenmeier, Simone Schmitz-Spanke Universitätsklinikum Essen, Hygiene und Arbeitsmedizine, Essen, Germany Introduction: Bladder cancer is the fifth most common malignancy in the world. Benzo(a)pyrene is among the 38 substances, believed to be responsible for bladder specific carcinogenic effect. However, the majority of studies on the toxicity of BaP are genotoxic. The aim of present study is to carry out the toxicoproteomic study of BaP with pig as the model organism. As the pig resembles humans in many physiological traits, pigs can be used as a valuable model organism for toxicological studies. Methodology: PUBECs (pig epithelial cell) were isolated from bladders of freshly slaughtered pigs. After washing, cells derived from different bladders were combined and resuspended in serum free culture medium (F-12 medium plus supplements; Guhe and Föllmann, 1994). 72 h cultured cells were exposed to 0.5 M BaP for another 24 h. Finally, the cells were harvested and subsequently lysed in lysis buffer. The supernatant was separated by 2D Gel electrophoresis and proteins were visualised by colloidal coomassie and analysed with 2D 3.6 (Decodon). Further, spots of interest were excised from the gels and identified by MALDI-ToF mass spectrometry. Conclusion: A total of 33 interesting proteins were identified. A further evaluation of identified proteins will help in identifying molecular mechanism of BaP toxicity. Because of the similarity to humans, proteomic studies on pigs will give opportunities to develop bladder cancer biomarkers.
Reference Guhe, C., Föllmann, W., 1994. Growth and chatacterization of porcine urinary bladder epithelial cells in vitro. Am. J. Physiol. 266, F298–F308.
doi:10.1016/j.toxlet.2009.06.587
E19 Identification of molecular biomarkers for endocrine disrupting compounds in Chironomus riparius (Diptera), a model species in aquatic ecotoxicology Gloria Morcillo ∗ , Rosario Planelló, Monica Morales, Estrella Cortés, Pedro M. de Paz, Jose Luis Martínez-Guitarte UNED, Biology and Environmental Toxicology, Madrid, Spain There is a growing list of chemicals with endocrine disruptor activity whose mechanisms of action have been well studied in humans and in a number of vertebrate models. Endocrinedisrupting chemicals (EDCs) can affect the synthesis or secretion of hormones and alter their endogenous functions. Although effects on reproduction and development have also been described in invertebrates, data at the molecular level are scarce. The midge Chironomus is widely used in aquatic toxicology and is considered to be an appropriate test species for research about potential endocrine disrupting substances. We have selected different compounds with known endocrine-disrupting activity in vertebrates to analyse their potential effect on the insect endocrine system. The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout development. The first target for its action inside the cells is the 20-OH ecdysone receptor, a heterodimer comprised of two elements, the ecdysone receptor (EcR) and the ultraspiracle (USP). RT-PCR was used to analyse the effects of acute and short time exposures to the selected compounds on the expression levels of these genes. The results show significant up-regulation of the ecdysone receptor (EcR) gene, with different susceptibility to each of the EDCs tested. These data provide, for the first time, a molecular mechanism for the action of these compounds in insects and suggest that the EcR gene could be a potential biomarker of exposure to compounds with activity as endocrine disruptors. Acknowledgement: Funded by CICYT, Spain, Project ref: CTM2006-12479. doi:10.1016/j.toxlet.2009.06.588
E20 Histopathological changes in the gills of freshwater fish, Oreochromis niloticus (L.) Juveniles exposed to Moringa oleifera (Lam.) fresh root-bark extract Babatunde Taiwo Adesina 1,∗ , A.O. Oguntuga 2 1
Osun State University, Animal Science & Fisheries, Osogbo, Nigeria, University of Ibadan, Department of Wildlife and Fisheries Management, Ibadan, Nigeria 2
Chronic sublethal toxicity of Moringa oleifera fresh root-bark extract on freshwater fish, Oreochromis niloticus juveniles was assessed using static renewable bioassay method to determine the toxicity of the extract on the gills of O. niloticus juveniles. However, Histopathology is a function of toxicity test to determine the safety
Abstracts / Toxicology Letters 189S (2009) S57–S273
level of exposure to toxicants in aquatic environment. Histopathological lesions in the gills of Oreochromis niloticus juveniles were assessed after 12-week exposure to fresh root-bark cold water extract of Moringa oleifera at chronic-sublethal concentrations of 0, 3 and 6 mg l−1 , where 0 mg l−1 served as the control group. Gills of O. niloticus juveniles exposed to 3 and 6 mg l−1 were collected and processed in an absolute ethanol along with the control group. They were later transferred and stained with haematoxylin and eosin according to the standard routine histological methods. Histopathological changes were recorded in the gills of Oreochromis niloticus juveniles exposed to chronic sublethal concentrations of M. oleifera fresh root-bark extract after 12-week exposure. After long exposure to the M. oleifera extract, major histopathological changes that occurred during the exposure were, gill lesions, gill arches vacuolation, lamellar hyperemia, complete degeneration of lamellae and reduction in the contents of all parts of gills. There were no lesions in gills of control fish as sign of histopathological changes. Moringa oleifera is toxic even at sublethal concentrations to freshwater fish, O. niloticus juveniles. However, it could be used as a risk assessment tool to establish safe limits to wipe out predatory fish in ponds prior to stocking. Keywords: Chronic-toxicity; Moringa oleifera; Oreochromis niloticus; Gill; Histology doi:10.1016/j.toxlet.2009.06.589
E21 ATHON: Studying toxicity and transport of highly purified NDLPCBs in a refined in vitro model of the human placental barrier Sara Correia Carreira 1,∗ , Laura Cartwright 1 , Margaret Saunders 1 , Susan Thawley 2 1
Bristol Haematology and Oncology Centre, Biophysics, Bristol, United Kingdom, 2 University of Bath, Department of Biology and Biochemistry, Bath, United Kingdom
Current findings suggest non-dioxin-like PCBs (NDL-PCBs) exert adverse effects on fetal development. Therefore, the objective of our work was to establish a robust in vitro model of the human placental barrier, in order to estimate the extent of potential hazard to the foetus. Here we present results on model development, toxicity, transport and accumulation of selected NDL-PCBs. We developed a robust model of the cytotrophoblast by growing monolayers of the human choriocarcinoma cell line BeWo on permeable Transwells® . Imaging and transport markers confirmed monolayer formation on day 4 post-seeding. Barrier integrity was further assessed by TEER measurements. A pre-determined TEER range formed the basis of a standard operating procedure developed for quality control. Cytotoxic effects of PCB52, PCB153 and PCB180 were determined by WST-1, LDH, and Alamar Blue assays. Due to interference of the PCBs with the WST-1 reagent, only LDH and Alamar Blue assays were used to assess toxicity. Results from the LDH assays indicate that the higher chlorinated PCBs induce toxicity at lower concentrations than PCB52. Preliminary analysis suggests the LDH assay is more sensitive than the Alamar Blue assay, therefore being better able to detect toxic effects of NDL-PCBs. Ongoing research in our lab includes measurement of cytokine expression after NDL-PCBs exposure to further investigate toxicity on the placental barrier. Our transport experiments showed that PCB52 and PCB180 have comparable permeability coefficients to Mannitol. However, more
S195
PCB52 and PCB180 than Mannitol associates to the monolayer, which may be due to the high lipid content in the cells. doi:10.1016/j.toxlet.2009.06.590
E22 The effect of different substances on Danio rerio and Poecilia reticulate Petra Doleˇzelová ∗ , Stanislava Mácová, Vladimíra Piˇstˇeková, ˇ Svobodová, Iveta Bedánová, ˇ Zdenka Eva Vosláˇrová University of Veterinary and Pharmaceutical Sciences, Department of Veterinary Public Health and Toxicology, Brno, Czech Republic Danio rerio and Poecilia reticulata are considered frequently used fish species when performing toxicity tests. This study assesses and compares sensitivity of these species to different type of substances usually found in water environment. One group of these substances is intended for treatment of fish (formaldehyde and potassium permanganate); the second group is presented in freshwater as pollutants (nitrite and silver nitrate). The semistatic method according to OECD No. 203 (Fish acute toxicity test) was obeyed for carrying out the tests. Both zebrafish and guppies were exposed in progressive concentrations series of each substance. In each test series, four acute toxicity tests were performed, with 10 fish used for each concentration and for the control group. The results (number of dead fish in individual test concentrations) were subjected to the probit analysis (EKO-TOX 5.1 software) to determine the 96hLC50 values. Statistical significance of the difference between LC50 values in guppies and zebrafish were evaluated by using the Unistat 5.1 programme. Regarding our findings, no difference in sensitivity was found between above mentioned fish species in formaldehyde, potassium permanganate and silver nitrate. However, the nitrate toxicity was significantly higher (p < 0.05) in Poecilia reticulata than in Danio rerio. Acknowledgement: The work was realized with the support of MSM Project No. 6215712402 Veterinary Aspects of Foodstuff Safety and Quality. doi:10.1016/j.toxlet.2009.06.591
E23 Comparison of the sensitivity of embryonic and juvenile developmental stages of Danio rerio to chemical substances Stanislava Mácová ∗ , Petra Doleˇzelová, Lucie Plhalová, Vladimíra ˇ Svobodová, Iveta Bedánová, ˇ Piˇstˇeková, Zdenka Eva Vosláˇrová University of Veterinary and Pharmaceutical Sciences, Brno, Department of Veterinary Public Health and Toxicology, Brno, Czech Republic The aim of this study is to compare sensitivity of embryonic and juvenile developmental stages of Danio rerio in acute toxicity test. Different chemical substances with another mode of action were selected. These chemicals are used in aquaculture or are presented in water environment (2-phenoxyethnol, clove oil, praziquantel, formaldehyde, potassium permanganate, PAX, nitrites, sodium chloride). The acute toxicity tests were performed according to OECD methods. In the acute toxicity tests with juvenile fish the
Abstracts / Toxicology Letters 189S (2009) S57–S273
and immune system. Little is understood regarding the specific mechanisms by which PCBs may exert their effects in biological systems. The in vitro BALB/c 3T3 cell transformation assay is a model resembling the in vivo multistep carcinogenesis and has being used for several years to screen the carcinogenicity of chemical and physical agents. We investigated Aroclor 1260 and observed a reduced cloning efficiency of 3T3 cells. The occurrence of morphologically transformed foci was related to the higher dose (20 M). The MTT assay was performed to evaluate the effects of the exposure to a wide range of concentrations (0.001–100 M) of A1260 and two single congeners, PCB 118 and PCB 153, on the proliferative rate of BALB/c 3T3 cells. A1260 induced a toxic effect higher then PCB118 and PCB 153. A global approach utilizing oligo-DNA microarray was used to assess cellular responses to low doses (5, 50 and 500 nM) of A1260 in BALB/c 3T3 cells and to identify genes that are transcriptionally regulated by PCBs exposure. The transcriptional profile makes it possible to analyze real mixtures extracted from environmental matrices and to identify the biomolecular markers directly related to exposure. Acknowledgement: Supported by “Fondazione del Monte di Bologna e Ravenna”, Bologna, Italy. doi:10.1016/j.toxlet.2009.06.586
E18 Pig as a model organism for toxicoproteomic studies on bladder cancer Nisha Verma ∗ , Albert W. Rettenmeier, Simone Schmitz-Spanke Universitätsklinikum Essen, Hygiene und Arbeitsmedizine, Essen, Germany Introduction: Bladder cancer is the fifth most common malignancy in the world. Benzo(a)pyrene is among the 38 substances, believed to be responsible for bladder specific carcinogenic effect. However, the majority of studies on the toxicity of BaP are genotoxic. The aim of present study is to carry out the toxicoproteomic study of BaP with pig as the model organism. As the pig resembles humans in many physiological traits, pigs can be used as a valuable model organism for toxicological studies. Methodology: PUBECs (pig epithelial cell) were isolated from bladders of freshly slaughtered pigs. After washing, cells derived from different bladders were combined and resuspended in serum free culture medium (F-12 medium plus supplements; Guhe and Föllmann, 1994). 72 h cultured cells were exposed to 0.5 M BaP for another 24 h. Finally, the cells were harvested and subsequently lysed in lysis buffer. The supernatant was separated by 2D Gel electrophoresis and proteins were visualised by colloidal coomassie and analysed with 2D 3.6 (Decodon). Further, spots of interest were excised from the gels and identified by MALDI-ToF mass spectrometry. Conclusion: A total of 33 interesting proteins were identified. A further evaluation of identified proteins will help in identifying molecular mechanism of BaP toxicity. Because of the similarity to humans, proteomic studies on pigs will give opportunities to develop bladder cancer biomarkers.
Reference Guhe, C., Föllmann, W., 1994. Growth and chatacterization of porcine urinary bladder epithelial cells in vitro. Am. J. Physiol. 266, F298–F308.
doi:10.1016/j.toxlet.2009.06.587
E19 Identification of molecular biomarkers for endocrine disrupting compounds in Chironomus riparius (Diptera), a model species in aquatic ecotoxicology Gloria Morcillo ∗ , Rosario Planelló, Monica Morales, Estrella Cortés, Pedro M. de Paz, Jose Luis Martínez-Guitarte UNED, Biology and Environmental Toxicology, Madrid, Spain There is a growing list of chemicals with endocrine disruptor activity whose mechanisms of action have been well studied in humans and in a number of vertebrate models. Endocrinedisrupting chemicals (EDCs) can affect the synthesis or secretion of hormones and alter their endogenous functions. Although effects on reproduction and development have also been described in invertebrates, data at the molecular level are scarce. The midge Chironomus is widely used in aquatic toxicology and is considered to be an appropriate test species for research about potential endocrine disrupting substances. We have selected different compounds with known endocrine-disrupting activity in vertebrates to analyse their potential effect on the insect endocrine system. The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout development. The first target for its action inside the cells is the 20-OH ecdysone receptor, a heterodimer comprised of two elements, the ecdysone receptor (EcR) and the ultraspiracle (USP). RT-PCR was used to analyse the effects of acute and short time exposures to the selected compounds on the expression levels of these genes. The results show significant up-regulation of the ecdysone receptor (EcR) gene, with different susceptibility to each of the EDCs tested. These data provide, for the first time, a molecular mechanism for the action of these compounds in insects and suggest that the EcR gene could be a potential biomarker of exposure to compounds with activity as endocrine disruptors. Acknowledgement: Funded by CICYT, Spain, Project ref: CTM2006-12479. doi:10.1016/j.toxlet.2009.06.588
E20 Histopathological changes in the gills of freshwater fish, Oreochromis niloticus (L.) Juveniles exposed to Moringa oleifera (Lam.) fresh root-bark extract Babatunde Taiwo Adesina 1,∗ , A.O. Oguntuga 2 1
Osun State University, Animal Science & Fisheries, Osogbo, Nigeria, University of Ibadan, Department of Wildlife and Fisheries Management, Ibadan, Nigeria 2
Chronic sublethal toxicity of Moringa oleifera fresh root-bark extract on freshwater fish, Oreochromis niloticus juveniles was assessed using static renewable bioassay method to determine the toxicity of the extract on the gills of O. niloticus juveniles. However, Histopathology is a function of toxicity test to determine the safety
Abstracts / Toxicology Letters 189S (2009) S57–S273
level of exposure to toxicants in aquatic environment. Histopathological lesions in the gills of Oreochromis niloticus juveniles were assessed after 12-week exposure to fresh root-bark cold water extract of Moringa oleifera at chronic-sublethal concentrations of 0, 3 and 6 mg l−1 , where 0 mg l−1 served as the control group. Gills of O. niloticus juveniles exposed to 3 and 6 mg l−1 were collected and processed in an absolute ethanol along with the control group. They were later transferred and stained with haematoxylin and eosin according to the standard routine histological methods. Histopathological changes were recorded in the gills of Oreochromis niloticus juveniles exposed to chronic sublethal concentrations of M. oleifera fresh root-bark extract after 12-week exposure. After long exposure to the M. oleifera extract, major histopathological changes that occurred during the exposure were, gill lesions, gill arches vacuolation, lamellar hyperemia, complete degeneration of lamellae and reduction in the contents of all parts of gills. There were no lesions in gills of control fish as sign of histopathological changes. Moringa oleifera is toxic even at sublethal concentrations to freshwater fish, O. niloticus juveniles. However, it could be used as a risk assessment tool to establish safe limits to wipe out predatory fish in ponds prior to stocking. Keywords: Chronic-toxicity; Moringa oleifera; Oreochromis niloticus; Gill; Histology doi:10.1016/j.toxlet.2009.06.589
E21 ATHON: Studying toxicity and transport of highly purified NDLPCBs in a refined in vitro model of the human placental barrier Sara Correia Carreira 1,∗ , Laura Cartwright 1 , Margaret Saunders 1 , Susan Thawley 2 1
Bristol Haematology and Oncology Centre, Biophysics, Bristol, United Kingdom, 2 University of Bath, Department of Biology and Biochemistry, Bath, United Kingdom
Current findings suggest non-dioxin-like PCBs (NDL-PCBs) exert adverse effects on fetal development. Therefore, the objective of our work was to establish a robust in vitro model of the human placental barrier, in order to estimate the extent of potential hazard to the foetus. Here we present results on model development, toxicity, transport and accumulation of selected NDL-PCBs. We developed a robust model of the cytotrophoblast by growing monolayers of the human choriocarcinoma cell line BeWo on permeable Transwells® . Imaging and transport markers confirmed monolayer formation on day 4 post-seeding. Barrier integrity was further assessed by TEER measurements. A pre-determined TEER range formed the basis of a standard operating procedure developed for quality control. Cytotoxic effects of PCB52, PCB153 and PCB180 were determined by WST-1, LDH, and Alamar Blue assays. Due to interference of the PCBs with the WST-1 reagent, only LDH and Alamar Blue assays were used to assess toxicity. Results from the LDH assays indicate that the higher chlorinated PCBs induce toxicity at lower concentrations than PCB52. Preliminary analysis suggests the LDH assay is more sensitive than the Alamar Blue assay, therefore being better able to detect toxic effects of NDL-PCBs. Ongoing research in our lab includes measurement of cytokine expression after NDL-PCBs exposure to further investigate toxicity on the placental barrier. Our transport experiments showed that PCB52 and PCB180 have comparable permeability coefficients to Mannitol. However, more
S195
PCB52 and PCB180 than Mannitol associates to the monolayer, which may be due to the high lipid content in the cells. doi:10.1016/j.toxlet.2009.06.590
E22 The effect of different substances on Danio rerio and Poecilia reticulate Petra Doleˇzelová ∗ , Stanislava Mácová, Vladimíra Piˇstˇeková, ˇ Svobodová, Iveta Bedánová, ˇ Zdenka Eva Vosláˇrová University of Veterinary and Pharmaceutical Sciences, Department of Veterinary Public Health and Toxicology, Brno, Czech Republic Danio rerio and Poecilia reticulata are considered frequently used fish species when performing toxicity tests. This study assesses and compares sensitivity of these species to different type of substances usually found in water environment. One group of these substances is intended for treatment of fish (formaldehyde and potassium permanganate); the second group is presented in freshwater as pollutants (nitrite and silver nitrate). The semistatic method according to OECD No. 203 (Fish acute toxicity test) was obeyed for carrying out the tests. Both zebrafish and guppies were exposed in progressive concentrations series of each substance. In each test series, four acute toxicity tests were performed, with 10 fish used for each concentration and for the control group. The results (number of dead fish in individual test concentrations) were subjected to the probit analysis (EKO-TOX 5.1 software) to determine the 96hLC50 values. Statistical significance of the difference between LC50 values in guppies and zebrafish were evaluated by using the Unistat 5.1 programme. Regarding our findings, no difference in sensitivity was found between above mentioned fish species in formaldehyde, potassium permanganate and silver nitrate. However, the nitrate toxicity was significantly higher (p < 0.05) in Poecilia reticulata than in Danio rerio. Acknowledgement: The work was realized with the support of MSM Project No. 6215712402 Veterinary Aspects of Foodstuff Safety and Quality. doi:10.1016/j.toxlet.2009.06.591
E23 Comparison of the sensitivity of embryonic and juvenile developmental stages of Danio rerio to chemical substances Stanislava Mácová ∗ , Petra Doleˇzelová, Lucie Plhalová, Vladimíra ˇ Svobodová, Iveta Bedánová, ˇ Piˇstˇeková, Zdenka Eva Vosláˇrová University of Veterinary and Pharmaceutical Sciences, Brno, Department of Veterinary Public Health and Toxicology, Brno, Czech Republic The aim of this study is to compare sensitivity of embryonic and juvenile developmental stages of Danio rerio in acute toxicity test. Different chemical substances with another mode of action were selected. These chemicals are used in aquaculture or are presented in water environment (2-phenoxyethnol, clove oil, praziquantel, formaldehyde, potassium permanganate, PAX, nitrites, sodium chloride). The acute toxicity tests were performed according to OECD methods. In the acute toxicity tests with juvenile fish the