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Cadmium in human pancreatic cancer – preliminary report
S92
Abstracts / Toxicology Letters 258S (2016) S62–S324
To analyze changes that occur upon treatment with the nongenotoxic carcinogen TCPOBOP, liver zonation and zonal effects were analyzed directly on the protein level. A time-series study with four groups of mice was performed and laser-capture micro dissection (LCM) was employed to isolate periportal and pericentral hepatocytes from mouse-liver sections. This material was analyzed for differences in expression and modification of more than 200 proteins and protein modifications using DigiWest, a novel and highly parallelized protein profiling technology. Local changes caused by the drug treatment were identified and modulation of activity of signal transduction pathways at the level of the zonal localization were observed. Pericentral hepatocytes respond differently to NGC treatment than periportal cells. Looking at individual zones instead of whole liver lysates leads to a refined understanding of early effects of phenobarbital-like NGCs. http://dx.doi.org/10.1016/j.toxlet.2016.06.1403 P03-003 Epigenetic silencing of the lung tumor suppressor cell adhesion molecule 1 J. Borlak 1,∗ , S.M. Reamon Buettner 2 1
Hannover Medical School, Hannover, Germany Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany 2
We previously reported the successful isolation and cultivation of spontaneously transformed cell lines from lung tumors of c-Myc & c-Raf transgenic mice. Oncogenomics revealed several tumor suppressors to be significantly repressed and included the cell adhesion molecule 1 (Cadm1) (Reamon-Buettner and Borlak, 2008). Its loss and/or reduction in activity correlate with poor prognosis in lung cancer patients. In pursue of mechanisms the Cadm1 gene regulation was investigated. Analysis of 69 CpGs displayed differential methylation pattern between and within tumor cell lines; apparently the degree of methylation correlated with transcriptional repression. DNA methylation analysis of the Cadm1 promoter revealed core CpGs in the binding sites of Sp1, Sp3 and zinc finger 5 to be hypermethylated, thus averting transcription factor (TF) binding. Alike, treatment of tumor cell lines with mithramycin A, an inhibitor of Sp1/Sp3 binding, resulted in reduction of Cadm1 gene expression to suggest a potential role of Sp1/Sp3 in Cadm1 gene regulation. Conversely, Cadm1 expression was restored by treatment of cell lines with the demethylating agent 5-aza-2 deoxycytidine. Subsequently performed single-molecule mapping with DNA methyltransferase M.SssI and micrococcal nuclease revealed high nucleosome occupancy and altered positioning (sliding) in silent Cadm1 promoters (Reamon-Buettner and Borlak, 2012). Additionally, chromatin immunoprecipitation of histone variants H2A.Z and H3.3 and H3K4me3 and H3K27me3 evidenced ‘bivalent’ histone modifications. Altogether, the studies highlight the complexity and different possibilities in the transcriptional repression of Cadm1 in lung cancer cells and broaden the perspective in developing epigenetic drugs. Reference Reamon-Buettner, Borlak, 2008. Cancer Res. 68, 7587–7596. Reamon-Buettner, Borlak, 2012. PLoS One 7 (6), e38531.
P03-004 Modification of histone H2b in a rat model by a reactive metabolite of the chemical carcinogen and food contaminant furan A.M. Antunes 1,∗ , J. Nunes 1 , I.L. Martins 1 , C. Charneira 1 , I. Pogribny 2 , A. De Conti 2 , F.A. Beland 2 , M. Marques 1 , C.C. Jacob 1 1
Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal 2 Division of Biochemical Toxicology, National Center for Toxicological Research (NCTR), Jefferson, AR 72079, USA
We report herein the first in vivo evidence for covalent modification of histone H2B by a reactive metabolite of the food contaminant and rat carcinogen, furan. We used a mass spectrometry-based bottom-up approach to identify a covalent lysine adduct in histones extracted from livers of male F344 rats treated by gavage with different doses of furan. The animal strain, dose and route of administration correspond to those used in a furan carcinogenicity 2-year bioassay, conducted by the NTP (1990). This covalent modification preceded the identification of altered epigenetic profiles, suggesting that it may take place at the early stages of furaninduced carcinogenesis. Moreover, it occurred in a lysine residue that is a target for epigenetic modifications and crucial for nucleosome stability. The formation of histone adducts may thus not only constitute accurate compound-specific biomarkers of exposure but also represent the missing link between exposure to carcinogens, aberrant epigenetic patterns, alteration of gene expression and nucleosome disassembly. Acknowledgements: This work was supported by a Cefic-LRI Innovative Science Award. We also acknowledge Fundac¸ão para a Ciência e a Tecnologia (FCT, Portugal) (grants RECI/QEQ-MED/0330/2012, IF/01091/2013/CP1163/CT0001, UID/QUI/00100/2013; fellowships SFRH/BD/75426/2010 to ILM and SFRH/BD/102846/2014 to CC). We thank the Portuguese MS network (IST node). AMMA also thanks Programa Operacional Potencial Humano from FCT and the European Social Fund (IF/01091/2013). Reference National Toxicology Program (NTP), 1990. Natl. Toxicol. Program Tech. Rep. Ser 382, 1–201.
http://dx.doi.org/10.1016/j.toxlet.2016.06.1405 P03-005 Cadmium in human pancreatic cancer – preliminary report V. Djordjevic 1 , D. Knezevic 1 , S. Matic 1 , M. Kerkez 1 , N. Zaric 1 , N. Grubor 1 , N. Bidzic 1 , N. Boricic 2 , I. Boricic 2 , V. Matovic 3 , A. Buha 3,∗ 1
Clinic for Digestive Surgery, First Surgical Clinic, Clinical Center of Serbia, Belgrade, Serbia 2 Department of Pathology, School of Medicine, University of Belgrade, Belgrade, Serbia 3 Department of Toxicology “Akademik Danilo Soldatovic”, University of Belgrade – Faculty of Pharmacy, Belgrade, Serbia
http://dx.doi.org/10.1016/j.toxlet.2016.06.1404 In the past decades, pancreatic cancer mortality gradually increased among both genders worldwide. The carcinogenic potential of cadmium (Cd), one of the most important environmental and occupational pollutants, has been approved by its classification as a
Abstracts / Toxicology Letters 258S (2016) S62–S324
S93
human carcinogen by IARC. Although Cd has been proven to induce lung cancer, state of the art investigations indicate that environmental Cd exposure could be associated with other types of cancer, pancreatic cancer being one of them. This study was aimed to determine Cd levels in pancreatic tissues of pancreatic cancer patients. Pancreatic tissue samples were obtained from 16 patients with pancreatic cancer of different stages, operated from May 2014 to May 2015. From each pancreatic tissue sample, cancer tissue and surrounding non-cancer tissue was collected. As controls, pancreatic tissue samples were taken during routine postmortem examinations from 13 fatalities of nonmalignant diseases. Cadmium levels were determined using graphite furnace atomic absorption following microwave assisted acid digestion of tissue samples. The mean Cd concentration determined in pancreatic cancer patients (10.33 ± 6.17 g/g) was significantly higher if compared to controls (1.98 ± 0.65 g/g). Furthermore, concentrations of Cd in cancer tissue was significantly elevated if compared with surrounding non-cancer tissue samples (p < 0.01). It could be concluded that malignant pancreatic tissue has the ability to accumulate Cd suggesting possible role of Cd in pancreatic cancer etiology. However, further investigations are needed in order to elucidate the hypothesis of Cd being one of the risk factors for pancreatic cancer development.
These models offer valuable information regarding molecular mechanistic insights of melanoma and could be further used for the assessment of antitumoral effects of different agents. This research was supported by the intern grant PII-C2-TC-201416498-10 offered by the “Victor Babes” University of Medicine and Pharmacy, Timisoara and by the PN-II-RU-TE-2014-4-2842 grant, no. 180/2015.
http://dx.doi.org/10.1016/j.toxlet.2016.06.1406
Purpose: Telomere is the repetition of the sequence 5 GGTTAG at the end of chromosome, which protects it from deterioration. Telomeres shortening can be influenced by the age, smoking, stressand dietary habits, leading at an increased cancer risk and heart diseases. Various studies show that the extension of telomere length, by improving DNA methylation pathway, can be achieved with folate, vitamins B12, D, A and K2, magnesium and omega3supplementation. The aim of this study is to determinate whether dietary vitamin supplements can influence telomeres shortening. Materials–methods: Metaphase spread leukocytes were isolated from peripheral blood. The telomere length measured by 3D Quantitative Fluorescence in situ Hybridization procedures (3D DNA FISH) with (C3TA2)3 peptide nucleic acid (PNA) probe. Photos from 10 metaphases from each individual were taken by Confocal microscopy and analyzed by image-J. Results and discussion: Thirty persons aged from 40 to 60-year old were included in this study. Eight of them were under treatment with vitamin supplements (VS group). No significant differences were observed in telomere length (kb) between participants’ sex (p = 0.168). The telomere length of VS group (mean telomere length: 11,730, median: 11,661) were significantly longer that those of the control groups (NVS group) (mean telomere length: 10,484, median: 10,287) (p = 0.046 and p = 0.027, respectively). Conclusion: This study reinforces the belief that the telomeres’ size can be increased with vitamin supplements. Perhaps a balanced diet adequate in vitamins could be the key for the protection from the telomeres shortening and relate chronic diseases.
http://dx.doi.org/10.1016/j.toxlet.2016.06.1407 P03-007 The luck of telomeres with or without the use of vitamin supplements E. Vakonaki 1 , P. Fragkiadaki 1 , E. Salataj 2,3 , A. Alegkakis 1 , E. Kouvidi 4 , M.N. Tzatzarakis 1 , C.G. Spilianakis 2,3 , A.M. Tsatsakis 1,∗ 1
Center of Toxicology Science and Research, University of Crete, Heraklion 71003, Greece 2 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology – Hellas, Heraklion 70013, Crete, Greece 3 Department of Biology, University of Crete, Heraklion, Crete, Greece 4 Department of Hematology, University of Crete School of Medicine, Heraklion, Greece
3
P03-006 Experimental models of human melanoma D.E. Coricovac 1,∗ , I. Pinzaru 1 , I.Z. Pavel 3 , R. Ghiulai 2 , A.M. Cimpean 4 , C. Soica 2 , C.A. Dehelean 1 , S. Avram 3 1 Department of Toxicology, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania 2 Department of Pharmaceutical Chemistry, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania 3 Department of Pharmacognosy, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania 4 Department of Histopathology, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania
Cutaneous malignant melanoma is one of the most violent forms of skin cancer with a high incidence and an increased rate of mortality. The therapeutic approaches existent are less efficient mainly when the metastasis occur. In order to find a curative treatment is mandatory to understand the molecular processes involved in the apparition, development and progression of melanoma. Based on these facts, we propose in the present study two reproducible experimental models of human melanoma using A375 – human melanoma xenografts. The first model is an animal model, using immunodeficient mice – Balb/c nude which were inoculated subcutaneously with 1X107 A375 cells/mouse. The second model consists of in shell A375 melanoma CAM (chorioallantoic membrane) assay, a fast and economic test. The animals developed amelanotic tumors starting with day 10–12 post-inoculation and at day 40 were detected spleen metastasis. At the end of experiment, the histopathological analysis indicated also, lung and liver metastasis. At day 4 post-inoculation of the cells on chorioallantoic membrane, there were noticed unpigmented tumors on the chorionic epithelium and progressive higher number of capillaries in spokes wheel around the cells. The histopathological analysis confirmed the presence of melanoma in the case of both models.
http://dx.doi.org/10.1016/j.toxlet.2016.06.1408
Abstracts / Toxicology Letters 258S (2016) S62–S324
To analyze changes that occur upon treatment with the nongenotoxic carcinogen TCPOBOP, liver zonation and zonal effects were analyzed directly on the protein level. A time-series study with four groups of mice was performed and laser-capture micro dissection (LCM) was employed to isolate periportal and pericentral hepatocytes from mouse-liver sections. This material was analyzed for differences in expression and modification of more than 200 proteins and protein modifications using DigiWest, a novel and highly parallelized protein profiling technology. Local changes caused by the drug treatment were identified and modulation of activity of signal transduction pathways at the level of the zonal localization were observed. Pericentral hepatocytes respond differently to NGC treatment than periportal cells. Looking at individual zones instead of whole liver lysates leads to a refined understanding of early effects of phenobarbital-like NGCs. http://dx.doi.org/10.1016/j.toxlet.2016.06.1403 P03-003 Epigenetic silencing of the lung tumor suppressor cell adhesion molecule 1 J. Borlak 1,∗ , S.M. Reamon Buettner 2 1
Hannover Medical School, Hannover, Germany Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany 2
We previously reported the successful isolation and cultivation of spontaneously transformed cell lines from lung tumors of c-Myc & c-Raf transgenic mice. Oncogenomics revealed several tumor suppressors to be significantly repressed and included the cell adhesion molecule 1 (Cadm1) (Reamon-Buettner and Borlak, 2008). Its loss and/or reduction in activity correlate with poor prognosis in lung cancer patients. In pursue of mechanisms the Cadm1 gene regulation was investigated. Analysis of 69 CpGs displayed differential methylation pattern between and within tumor cell lines; apparently the degree of methylation correlated with transcriptional repression. DNA methylation analysis of the Cadm1 promoter revealed core CpGs in the binding sites of Sp1, Sp3 and zinc finger 5 to be hypermethylated, thus averting transcription factor (TF) binding. Alike, treatment of tumor cell lines with mithramycin A, an inhibitor of Sp1/Sp3 binding, resulted in reduction of Cadm1 gene expression to suggest a potential role of Sp1/Sp3 in Cadm1 gene regulation. Conversely, Cadm1 expression was restored by treatment of cell lines with the demethylating agent 5-aza-2 deoxycytidine. Subsequently performed single-molecule mapping with DNA methyltransferase M.SssI and micrococcal nuclease revealed high nucleosome occupancy and altered positioning (sliding) in silent Cadm1 promoters (Reamon-Buettner and Borlak, 2012). Additionally, chromatin immunoprecipitation of histone variants H2A.Z and H3.3 and H3K4me3 and H3K27me3 evidenced ‘bivalent’ histone modifications. Altogether, the studies highlight the complexity and different possibilities in the transcriptional repression of Cadm1 in lung cancer cells and broaden the perspective in developing epigenetic drugs. Reference Reamon-Buettner, Borlak, 2008. Cancer Res. 68, 7587–7596. Reamon-Buettner, Borlak, 2012. PLoS One 7 (6), e38531.
P03-004 Modification of histone H2b in a rat model by a reactive metabolite of the chemical carcinogen and food contaminant furan A.M. Antunes 1,∗ , J. Nunes 1 , I.L. Martins 1 , C. Charneira 1 , I. Pogribny 2 , A. De Conti 2 , F.A. Beland 2 , M. Marques 1 , C.C. Jacob 1 1
Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal 2 Division of Biochemical Toxicology, National Center for Toxicological Research (NCTR), Jefferson, AR 72079, USA
We report herein the first in vivo evidence for covalent modification of histone H2B by a reactive metabolite of the food contaminant and rat carcinogen, furan. We used a mass spectrometry-based bottom-up approach to identify a covalent lysine adduct in histones extracted from livers of male F344 rats treated by gavage with different doses of furan. The animal strain, dose and route of administration correspond to those used in a furan carcinogenicity 2-year bioassay, conducted by the NTP (1990). This covalent modification preceded the identification of altered epigenetic profiles, suggesting that it may take place at the early stages of furaninduced carcinogenesis. Moreover, it occurred in a lysine residue that is a target for epigenetic modifications and crucial for nucleosome stability. The formation of histone adducts may thus not only constitute accurate compound-specific biomarkers of exposure but also represent the missing link between exposure to carcinogens, aberrant epigenetic patterns, alteration of gene expression and nucleosome disassembly. Acknowledgements: This work was supported by a Cefic-LRI Innovative Science Award. We also acknowledge Fundac¸ão para a Ciência e a Tecnologia (FCT, Portugal) (grants RECI/QEQ-MED/0330/2012, IF/01091/2013/CP1163/CT0001, UID/QUI/00100/2013; fellowships SFRH/BD/75426/2010 to ILM and SFRH/BD/102846/2014 to CC). We thank the Portuguese MS network (IST node). AMMA also thanks Programa Operacional Potencial Humano from FCT and the European Social Fund (IF/01091/2013). Reference National Toxicology Program (NTP), 1990. Natl. Toxicol. Program Tech. Rep. Ser 382, 1–201.
http://dx.doi.org/10.1016/j.toxlet.2016.06.1405 P03-005 Cadmium in human pancreatic cancer – preliminary report V. Djordjevic 1 , D. Knezevic 1 , S. Matic 1 , M. Kerkez 1 , N. Zaric 1 , N. Grubor 1 , N. Bidzic 1 , N. Boricic 2 , I. Boricic 2 , V. Matovic 3 , A. Buha 3,∗ 1
Clinic for Digestive Surgery, First Surgical Clinic, Clinical Center of Serbia, Belgrade, Serbia 2 Department of Pathology, School of Medicine, University of Belgrade, Belgrade, Serbia 3 Department of Toxicology “Akademik Danilo Soldatovic”, University of Belgrade – Faculty of Pharmacy, Belgrade, Serbia
http://dx.doi.org/10.1016/j.toxlet.2016.06.1404 In the past decades, pancreatic cancer mortality gradually increased among both genders worldwide. The carcinogenic potential of cadmium (Cd), one of the most important environmental and occupational pollutants, has been approved by its classification as a
Abstracts / Toxicology Letters 258S (2016) S62–S324
S93
human carcinogen by IARC. Although Cd has been proven to induce lung cancer, state of the art investigations indicate that environmental Cd exposure could be associated with other types of cancer, pancreatic cancer being one of them. This study was aimed to determine Cd levels in pancreatic tissues of pancreatic cancer patients. Pancreatic tissue samples were obtained from 16 patients with pancreatic cancer of different stages, operated from May 2014 to May 2015. From each pancreatic tissue sample, cancer tissue and surrounding non-cancer tissue was collected. As controls, pancreatic tissue samples were taken during routine postmortem examinations from 13 fatalities of nonmalignant diseases. Cadmium levels were determined using graphite furnace atomic absorption following microwave assisted acid digestion of tissue samples. The mean Cd concentration determined in pancreatic cancer patients (10.33 ± 6.17 g/g) was significantly higher if compared to controls (1.98 ± 0.65 g/g). Furthermore, concentrations of Cd in cancer tissue was significantly elevated if compared with surrounding non-cancer tissue samples (p < 0.01). It could be concluded that malignant pancreatic tissue has the ability to accumulate Cd suggesting possible role of Cd in pancreatic cancer etiology. However, further investigations are needed in order to elucidate the hypothesis of Cd being one of the risk factors for pancreatic cancer development.
These models offer valuable information regarding molecular mechanistic insights of melanoma and could be further used for the assessment of antitumoral effects of different agents. This research was supported by the intern grant PII-C2-TC-201416498-10 offered by the “Victor Babes” University of Medicine and Pharmacy, Timisoara and by the PN-II-RU-TE-2014-4-2842 grant, no. 180/2015.
http://dx.doi.org/10.1016/j.toxlet.2016.06.1406
Purpose: Telomere is the repetition of the sequence 5 GGTTAG at the end of chromosome, which protects it from deterioration. Telomeres shortening can be influenced by the age, smoking, stressand dietary habits, leading at an increased cancer risk and heart diseases. Various studies show that the extension of telomere length, by improving DNA methylation pathway, can be achieved with folate, vitamins B12, D, A and K2, magnesium and omega3supplementation. The aim of this study is to determinate whether dietary vitamin supplements can influence telomeres shortening. Materials–methods: Metaphase spread leukocytes were isolated from peripheral blood. The telomere length measured by 3D Quantitative Fluorescence in situ Hybridization procedures (3D DNA FISH) with (C3TA2)3 peptide nucleic acid (PNA) probe. Photos from 10 metaphases from each individual were taken by Confocal microscopy and analyzed by image-J. Results and discussion: Thirty persons aged from 40 to 60-year old were included in this study. Eight of them were under treatment with vitamin supplements (VS group). No significant differences were observed in telomere length (kb) between participants’ sex (p = 0.168). The telomere length of VS group (mean telomere length: 11,730, median: 11,661) were significantly longer that those of the control groups (NVS group) (mean telomere length: 10,484, median: 10,287) (p = 0.046 and p = 0.027, respectively). Conclusion: This study reinforces the belief that the telomeres’ size can be increased with vitamin supplements. Perhaps a balanced diet adequate in vitamins could be the key for the protection from the telomeres shortening and relate chronic diseases.
http://dx.doi.org/10.1016/j.toxlet.2016.06.1407 P03-007 The luck of telomeres with or without the use of vitamin supplements E. Vakonaki 1 , P. Fragkiadaki 1 , E. Salataj 2,3 , A. Alegkakis 1 , E. Kouvidi 4 , M.N. Tzatzarakis 1 , C.G. Spilianakis 2,3 , A.M. Tsatsakis 1,∗ 1
Center of Toxicology Science and Research, University of Crete, Heraklion 71003, Greece 2 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology – Hellas, Heraklion 70013, Crete, Greece 3 Department of Biology, University of Crete, Heraklion, Crete, Greece 4 Department of Hematology, University of Crete School of Medicine, Heraklion, Greece
3
P03-006 Experimental models of human melanoma D.E. Coricovac 1,∗ , I. Pinzaru 1 , I.Z. Pavel 3 , R. Ghiulai 2 , A.M. Cimpean 4 , C. Soica 2 , C.A. Dehelean 1 , S. Avram 3 1 Department of Toxicology, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania 2 Department of Pharmaceutical Chemistry, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania 3 Department of Pharmacognosy, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania 4 Department of Histopathology, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania
Cutaneous malignant melanoma is one of the most violent forms of skin cancer with a high incidence and an increased rate of mortality. The therapeutic approaches existent are less efficient mainly when the metastasis occur. In order to find a curative treatment is mandatory to understand the molecular processes involved in the apparition, development and progression of melanoma. Based on these facts, we propose in the present study two reproducible experimental models of human melanoma using A375 – human melanoma xenografts. The first model is an animal model, using immunodeficient mice – Balb/c nude which were inoculated subcutaneously with 1X107 A375 cells/mouse. The second model consists of in shell A375 melanoma CAM (chorioallantoic membrane) assay, a fast and economic test. The animals developed amelanotic tumors starting with day 10–12 post-inoculation and at day 40 were detected spleen metastasis. At the end of experiment, the histopathological analysis indicated also, lung and liver metastasis. At day 4 post-inoculation of the cells on chorioallantoic membrane, there were noticed unpigmented tumors on the chorionic epithelium and progressive higher number of capillaries in spokes wheel around the cells. The histopathological analysis confirmed the presence of melanoma in the case of both models.
http://dx.doi.org/10.1016/j.toxlet.2016.06.1408