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Transcriptome analysis of contact-inhibition
S134
Abstracts / Toxicology Letters 189S (2009) S57–S273
show that HCB increases the number and malignancy of tumors in NMU-induced mammary tumors in rats. c-Src and HER1 synergize to enhance neoplastic growth of mammary epithelial cells. This interaction is dependent on c-Src-mediated phosphorylation of Y845-HER1, triggering proliferation, migration and metastasis. c-Src activates estrogen receptor ␣ (ER␣) via Y537 phosphorylation. Our aim was to study HCB mechanism of action in mammary gland (M) and NMU-induced mammary tumors (T) in rats on: (a) HER1 levels and Y845-HER1 phosphorylation, (b) c-Src activation, (c) ER␣ levels and Y537-ER␣ phosphorylation, (d) ERK1/2 activation and (e) estrogen and progesterone serum levels. Study groups were: Control, HCB (100 mg/kg for 45 days), NMU (3 doses of 50 mg/kg) and NMU-HCB. Protein expression and phosphorylation were measured by immunoprecipitation and Western blot, and hormone levels by RIA. Results show that HCB: (a) enhanced Y845-HER1 phosphorylation in M and T; (b) increased c-Src protein levels and activation in M and T; (c) raised cytosolic and nuclear ER␣ levels in T; and enhanced Y537-ER␣ phosphorylation in M and decreased it in T; (d) elicited ERK1/2 activation in M and decreased it in T, (e) increased estrogen and decreased progesterone serum levels in HCB group, and conversely, decreased estrogen and increased progesterone serum levels in NMU-HCB treated rats. These results indicate that HCB induces c-Src dependent HER1 signaling pathway in mammary gland and tumors, which could explain the ductal hyperplasia and enhanced proliferation in mammary gland, and the increase in malignancy of tumors previously reported. doi:10.1016/j.toxlet.2009.06.716
A15 Low levels of arsenic enhance the genotoxicity induced by ochratoxin A in human urothelial (5637) cells M. Aggarwal 1,∗ , C. Behm 2 , W. Foellmann 2 , S.B. Dorn 3 , J.K. Malik 4 , G.H. Degen 2 FORIM GmbH, Toxicology, Mannheim, Germany, 2 Leibniz Research Centre for Working Environment and Human Factors (IfADo), Toxicology, Dortmund, Germany, 3 DR. KNOELL CONSULT GmbH, Toxicology, Leverkusen, Germany, 4 Indian Veterinary Research Institute, Division of Pharmacology and Toxicology, Izatnagar, India
1
Ochratoxin A (OTA) occurs worldwide in many food and feed commodities. The mycotoxin is a potent carcinogen in rats, with kidney and urinary bladder as major target organs, and is suspected to be involved in Balkan endemic nephropathy in humans. Arsenic, a potent carcinogen with multiple target organs including urinary bladder and kidney, is found in ground water in many parts of the world. As co-exposure to both contaminants is possible, their combinatory toxic effects raise concern. OTA and arsenic exert cytotoxicity in human urothelial (5637) cells with IC50 values of 8 and 4 M, respectively, for 48 h exposure. The effect is additive at most when tested in combination at concentrations of individual compounds that are toxic, but not at lower levels. Cytotoxic OTA levels caused cell cycle arrest at G2/M phase and apoptosis. Arsenic at 1 M did not cause such changes, but reduced the apoptotic cell fraction induced by OTA. OTA at 7.5 and 15 M caused induction of micronuclei (MN) after 30 h treatment; the MN were predominantly CREST-negative indicative of clastogenic effects. Arsenic alone at 1 M did not induce MN, but enhanced the OTA induced MN frequency in co-treatments. In single cell gel electrophoresis (Comet) assays, 10 M arsenic did not cause measurable DNA dam-
age after 3 h treatment, but it increased the damage induced by 100 M OTA. These data warrant further studies on interaction of OTA and arsenic. doi:10.1016/j.toxlet.2009.06.717
A16 Transcriptome analysis of contact-inhibition Cornelia Dietrich ∗ , Carina Ittrich, Firas Al-Butmeh, Dagmar Faust, Monika Küppers Universitätsmedizin Mainz, Institute of Toxicology, Mainz, Germany Purpose: Proliferation of non-transformed cells is regulated by cell–cell contacts, which are referred to as contact-inhibition. Vice versa, transformed cells are characterized by a loss of contactinhibition. Despite its generally accepted importance for cell cycle control, little is known about the intracellular signalling pathways involved in contact-inhibition. Understanding the molecular mechanisms of contact-inhibition and its loss during tumourigenesis will be an important step towards the identification of novel target genes in tumour treatment. Methods: We therefore identified the transcriptional programme of contact-inhibition in NIH3T3 fibroblast using highdensity microarrays. Results: We found 111 transcripts to be differentially expressed in confluent compared to exponentially growing cultures (cut off: ≥2-fold, p ≤ 0.002) representing 95 genes and 16 cDNA sequences involved, e.g. in proliferation, signal transduction, transcriptional regulation, cell adhesion and communication. Interestingly, the majority of genes was upregulated indicating that contactinhibition is not a passive state, but actively induced. 717 genes and 210 cDNA sequences were differentially expressed setting the cut off: ≥1.5-fold, p ≤ 0.05. Importing these data into GenMAPP software revealed for the first time a comprehensive list of cell cycle regulatory genes mediating G1-arrest, e.g. Skp2, and inhibition of DNA-synthesis, such as several Orc and Mcm proteins. Furthermore, using RT-PCR and Western blot, we identified the transcription factor FoxM1 as a novel gene to be downregulated in contact-inhibited cells. doi:10.1016/j.toxlet.2009.06.718
A17 The effects of curcumin as a chemoprotective agent against aflatoxin B1 induced toxicity in rats Amnart Poapolathep 1,∗ , Saranya Poapolathep 2 , Kenji Machii 3 , Hiroyuki Nakayama 4 , Susumu Kumagai 2 1
Kasetsart University, Department of Pharmacology, Faculty of Veterinary Medicine, Bangkok, Thailand, 2 University of Tokyo, Department of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, Tokyo, Japan, 3 National Institute of Health Sciences, Division of Biomedical Food Research, Tokyo, Japan, 4 University of Tokyo, Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, Tokyo, Japan Curcumin is a polyphenol compound which has wide range pharmacological activities. This compound is considered to be one of the most studied chemopreventive agents. The present study was designed to evaluate the effect of curcumin on aflatoxin B1 (AFB1) metabolism, its acute toxicity and its-induced hepatocarcinogene-
Abstracts / Toxicology Letters 189S (2009) S57–S273
show that HCB increases the number and malignancy of tumors in NMU-induced mammary tumors in rats. c-Src and HER1 synergize to enhance neoplastic growth of mammary epithelial cells. This interaction is dependent on c-Src-mediated phosphorylation of Y845-HER1, triggering proliferation, migration and metastasis. c-Src activates estrogen receptor ␣ (ER␣) via Y537 phosphorylation. Our aim was to study HCB mechanism of action in mammary gland (M) and NMU-induced mammary tumors (T) in rats on: (a) HER1 levels and Y845-HER1 phosphorylation, (b) c-Src activation, (c) ER␣ levels and Y537-ER␣ phosphorylation, (d) ERK1/2 activation and (e) estrogen and progesterone serum levels. Study groups were: Control, HCB (100 mg/kg for 45 days), NMU (3 doses of 50 mg/kg) and NMU-HCB. Protein expression and phosphorylation were measured by immunoprecipitation and Western blot, and hormone levels by RIA. Results show that HCB: (a) enhanced Y845-HER1 phosphorylation in M and T; (b) increased c-Src protein levels and activation in M and T; (c) raised cytosolic and nuclear ER␣ levels in T; and enhanced Y537-ER␣ phosphorylation in M and decreased it in T; (d) elicited ERK1/2 activation in M and decreased it in T, (e) increased estrogen and decreased progesterone serum levels in HCB group, and conversely, decreased estrogen and increased progesterone serum levels in NMU-HCB treated rats. These results indicate that HCB induces c-Src dependent HER1 signaling pathway in mammary gland and tumors, which could explain the ductal hyperplasia and enhanced proliferation in mammary gland, and the increase in malignancy of tumors previously reported. doi:10.1016/j.toxlet.2009.06.716
A15 Low levels of arsenic enhance the genotoxicity induced by ochratoxin A in human urothelial (5637) cells M. Aggarwal 1,∗ , C. Behm 2 , W. Foellmann 2 , S.B. Dorn 3 , J.K. Malik 4 , G.H. Degen 2 FORIM GmbH, Toxicology, Mannheim, Germany, 2 Leibniz Research Centre for Working Environment and Human Factors (IfADo), Toxicology, Dortmund, Germany, 3 DR. KNOELL CONSULT GmbH, Toxicology, Leverkusen, Germany, 4 Indian Veterinary Research Institute, Division of Pharmacology and Toxicology, Izatnagar, India
1
Ochratoxin A (OTA) occurs worldwide in many food and feed commodities. The mycotoxin is a potent carcinogen in rats, with kidney and urinary bladder as major target organs, and is suspected to be involved in Balkan endemic nephropathy in humans. Arsenic, a potent carcinogen with multiple target organs including urinary bladder and kidney, is found in ground water in many parts of the world. As co-exposure to both contaminants is possible, their combinatory toxic effects raise concern. OTA and arsenic exert cytotoxicity in human urothelial (5637) cells with IC50 values of 8 and 4 M, respectively, for 48 h exposure. The effect is additive at most when tested in combination at concentrations of individual compounds that are toxic, but not at lower levels. Cytotoxic OTA levels caused cell cycle arrest at G2/M phase and apoptosis. Arsenic at 1 M did not cause such changes, but reduced the apoptotic cell fraction induced by OTA. OTA at 7.5 and 15 M caused induction of micronuclei (MN) after 30 h treatment; the MN were predominantly CREST-negative indicative of clastogenic effects. Arsenic alone at 1 M did not induce MN, but enhanced the OTA induced MN frequency in co-treatments. In single cell gel electrophoresis (Comet) assays, 10 M arsenic did not cause measurable DNA dam-
age after 3 h treatment, but it increased the damage induced by 100 M OTA. These data warrant further studies on interaction of OTA and arsenic. doi:10.1016/j.toxlet.2009.06.717
A16 Transcriptome analysis of contact-inhibition Cornelia Dietrich ∗ , Carina Ittrich, Firas Al-Butmeh, Dagmar Faust, Monika Küppers Universitätsmedizin Mainz, Institute of Toxicology, Mainz, Germany Purpose: Proliferation of non-transformed cells is regulated by cell–cell contacts, which are referred to as contact-inhibition. Vice versa, transformed cells are characterized by a loss of contactinhibition. Despite its generally accepted importance for cell cycle control, little is known about the intracellular signalling pathways involved in contact-inhibition. Understanding the molecular mechanisms of contact-inhibition and its loss during tumourigenesis will be an important step towards the identification of novel target genes in tumour treatment. Methods: We therefore identified the transcriptional programme of contact-inhibition in NIH3T3 fibroblast using highdensity microarrays. Results: We found 111 transcripts to be differentially expressed in confluent compared to exponentially growing cultures (cut off: ≥2-fold, p ≤ 0.002) representing 95 genes and 16 cDNA sequences involved, e.g. in proliferation, signal transduction, transcriptional regulation, cell adhesion and communication. Interestingly, the majority of genes was upregulated indicating that contactinhibition is not a passive state, but actively induced. 717 genes and 210 cDNA sequences were differentially expressed setting the cut off: ≥1.5-fold, p ≤ 0.05. Importing these data into GenMAPP software revealed for the first time a comprehensive list of cell cycle regulatory genes mediating G1-arrest, e.g. Skp2, and inhibition of DNA-synthesis, such as several Orc and Mcm proteins. Furthermore, using RT-PCR and Western blot, we identified the transcription factor FoxM1 as a novel gene to be downregulated in contact-inhibited cells. doi:10.1016/j.toxlet.2009.06.718
A17 The effects of curcumin as a chemoprotective agent against aflatoxin B1 induced toxicity in rats Amnart Poapolathep 1,∗ , Saranya Poapolathep 2 , Kenji Machii 3 , Hiroyuki Nakayama 4 , Susumu Kumagai 2 1
Kasetsart University, Department of Pharmacology, Faculty of Veterinary Medicine, Bangkok, Thailand, 2 University of Tokyo, Department of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, Tokyo, Japan, 3 National Institute of Health Sciences, Division of Biomedical Food Research, Tokyo, Japan, 4 University of Tokyo, Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, Tokyo, Japan Curcumin is a polyphenol compound which has wide range pharmacological activities. This compound is considered to be one of the most studied chemopreventive agents. The present study was designed to evaluate the effect of curcumin on aflatoxin B1 (AFB1) metabolism, its acute toxicity and its-induced hepatocarcinogene-