evaluated, Cul4a was the only one statistically affected by thalidomide in the first 24hs, having its expression diminished. The whole complex (Crbn, Ddb1, ...
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Abstracts / Reproductive Toxicology 72 (2017) 22–27
Secretoglobin 1A1 (SCGB1A1) protein expressed immunohistochemically in neonatal mice lung Club cells after exposure at heavy traffic sites in Northern Greece Katerina Kaidoglou a,∗ , Maria Bousnaki a , Constantini Samara b , Elpida-Niki Emmanouil-Nikoloussi a,c,d a
Laboratory of Histology-Embryology and Anthropology, Faculty of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece b Enviromental Pollution Control Laboratory, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece c Environmental Department, Municipality of Thessaloniki, Greece d School of Medicine, European University Cyprus, Nicosia, Cyprus Introduction: Particulate matter (PM) of the ambient air resulting from erosion, engines combustion and diesel exhaust, are associated with increased incidence of respiratory diseases. Epidemiological studies demonstrated association between fine particles, lung cancer and different cardiopulmonary diseases. Secretoglobin 1A1 (SCGB1A1) protein is a member of the secretoglobin family, specifically expressed in the non-ciliated lung Club cells (formerly “Clara”). Their secretion contributes to the bronchiolar fluid reducing surface tension. Serum club cell protein-CC16 was found decreased in several occupational groups chronically exposed to different air pollutants. Association between ambient air pollutants and serum concentration of CC16 protein supports the idea that ambient PM promotes respiratory diseases increasing lungs’ epithelial barrier permeability [1–3]. Methods: Investigation by immunohistochemistry PM effects on Secretoglobin 1A1 (SCGB1A1) protein production, into Club cells of neonate breastfeeding mice, exposed at the center of an urban area of Thessaloniki, a heavy traffic city, in Northern Greece. Fifteen female Balb/c mice positive for vaginal plugs were considered to be at gestational day 0. They were divided into two groups, study group: 10 mice and control group: 5 mice. Animals were placed in the Municipality’s of Thessaloniki Air Quality Monitoring Station, located at the city center, where “study” group was exposed to atmospheric air and “control” group to filtered air. During exposure, animals had access to food and water “ad libitum”. After delivery, 20 breastfeeding neonates (study group) were exposed to atmospheric air, while 5 control breastfeeding neonates were exposed to filtered air. After 20 days exposure, neonates were sacrificed by ether lethal doses. Neonatal lung tissue samples were collected and using a polyclonal antibody, Secretoglobin 1A1 were detected with commercially supplied reagents. Results: Club cells in alveolar and bronchiole epithelium of neonatal mice were positive to Secretoglobin 1A1 polyclonal antibody at a multidimensional grade. This marker can be used as a detector of an acute or chronic disruption of the bronchoalveolar/blood barrier integrity. Conclusion: Despite constantly conducted large-scale studies involving both physiological and pathological conditions, the role of Club cells is still not fully understood. PM and pollutants in general are influencing their function in neonate breastfeeding mice increasing their Secretoglobin 1A1 (SCGB1A1) protein secretion during acute exposure to pollutants, possibly as a mechanism of their defense to them.
Acknowledgements This research has been co-financed by the European Union (European Social Fund – ESF) and the Greek Ministry of Education through the Research Funding Program 409 THALES (Project code/Title: MIS 377304/“Bioactivity of airborne particulates in relation with their size, morphology and chemical composition”). References [1] B.R. Celli, C.A. Owen, The club cell and its protein, CC16: time to shine, Lancet Respir. Med. 1 (10) (2013) 757–759. [2] C. Hermans, A. Bernard, Clara cell protein (CC16): characteristics and potential applications as biomarker of lung toxicity, Biomarkers 1 (1) (2008) 3–8. [3] C. Madsen, Associations between ambient air pollution and serum Clara cell protein concentrations, Epidemiology 18 (5) (2007) 106.
http://dx.doi.org/10.1016/j.reprotox.2017.06.060 The role of metabolism in the prenatal developmental toxicity (PDT) of polycyclic aromatic hydrocarbons (PAHs) in petroleum substances Lenny Kamelia a,∗ , Jochem Louisse a , Ivonne M.C.M. Rietjens a , Peter J. Boogaard a,b a
Division of Toxicology, Wageningen University, The Netherlands b Shell Health, Shell International bv, The Hague, The Netherlands Recently we reported on the use of embryonic stem cell test (EST) to study PDT of petroleum substances (PS) and gas-to-liquid (GTL) products [1]. A highly significant correlation was found between the EST results and in vivo data. Additionally, the results in the EST correlated very well with the concentration of certain PAHs in the tested substances. This is surprising since it is generally assumed that PAHs require metabolism to exert their adverse effects. For instance, benzo(a)pyrene (BaP) is embryotoxic in vivo but not in in vitro assays without a metabolic system [2]. To better mimic the in vivo situation and to see if the toxicity potency changes with biotransformation, the inclusion of a metabolic system in in vitro assays is highly relevant. Here we combined a metabolism system with the EST to compare the in vitro PDT potencies of substances with and without the presence of biotransformation enzymes. The metabolite mixtures of the test substances were generated using hamster liver microsomes. Since it is known that S9 mix/liver microsomes are toxic for embryonic stem cells [3], the incubation mixtures containing the metabolite cocktails were extracted using diisopropyl ether, evaporated, and re-dissolved in DMSO before application in the EST. The following samples were tested in the EST, both with and without metabolic activation: several individual PAHs, five DMSO-extracts of PS, and one of a GTL-base oil (GTLb). The results show that BaP does not inhibit the differentiation of ES-D3 cells into contracting cardiomyocytes up to the highest tested concentration (12.6 g/ml), whereas dibenz(a,h)anthracene (DBA) inhibits the ES-D3 cell differentiation in a concentration-dependent manner (BMCd 50:3 g/ml). Remarkably, the bioactivated BaP induced a concentration-dependent inhibition of ES-D3 cell differentiation (BMCd 50:1 g/ml). This indicates that BaP needs to be transformed into its reactive metabolites, including 3-hydroxybenzo(a)pyrene (which was shown to be active in the EST itself), to exert its embryotoxic effect. Subsequently, we used the same approach for the PS and GTLb samples, and their potencies in the EST, with and without metabolism, was compared. From our previous study [1], the DMSO-extracts of PS were able to inhibit the differentiation of
Abstracts / Reproductive Toxicology 72 (2017) 22–27
ES-D3 cells. It is interesting to see that those potencies were not notably changed when ES-D3 cells were exposed to the metabolite mixtures of the corresponding PS. This suggests that the metabolism does not play a crucial role for PS to exert their in vitro embryotoxicity effects. Further, there are some constituents of PS, such as DBA, that are capable to induce PDT without any biotransformation. In other words, there are some substances that are active without bioactivation and are able to interact with the cellular components inducing embryotoxicity. To conclude, a biotransformation system was well-incorporated to the EST, and that the PDT potency of some substances has changed after the biotransformation due to the formation of reactive metabolites. Also, some of the PAHs in PS are able to induce PDT without biotransformation.
References [1] L. Kamelia, J. Louisse, I.M.C.M. Rietjens, P.J. Boogaard,. Prenatal developmental toxicity testing of petroleum substances: application of the mouse embryonic stem cell test (EST) to compare in vitro potencies with potencies observed in vivo (submitted for publication). [2] A. Langsch, H. Nau, Metabolic activation for in vitro systems, ALTEX 22 (Special Issue 2) (2005) 354–358. [3] A.M. Wobus, P. Löser, Present state and future perspectives of using pluripotent stem cells in toxicology research, Arch. Toxicol. 85 (2011) 79–117.
http://dx.doi.org/10.1016/j.reprotox.2017.06.061 Gene expression changes after thalidomide exposure in different species: Preliminary results regarding Cereblon complex and angiogenesis components Thayne W. Kowalski a,b,c,∗ , Lucas R. Fraga a,b,c , Julia A. Gomes a,b,c , Lavinia Schuler-Faccini a,b,c , Fernanda S.L. Vianna a,b,c
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thalidomide in the first 24hs, having its expression diminished. The whole complex (Crbn, Ddb1, Cul4a and Rbx1) is statistically affected by the drug at 48 h; Ddb1 is the only with increased expression after the exposure. At 72 h, only Nos3 and Vegfa are statistically different in control versus exposed cells, having a lower expression in the ones that received thalidomide. Conclusions: These results are part of a larger aim that intends to evaluate the differences in gene expression after thalidomide exposure across a variety of species. Although mice are less sensitive to thalidomide, this drug could affect the expression of Cereblon complex and angiogenesis components, as previously identified in more sensitive species. Apparently, this effect occurs first in the Cereblon complex proteins (especially in Cul4a), and then in the components of the angiogenesis pathway here evaluated. We also observed an increase in the expression of Ddb1 gene. Other studies are necessary to confirm or refute the data here presented. As perspective, we intend to evaluate similar data of other species, such as rats, chickens, monkeys and humans. We believe thalidomide’s effects in genomic expression across different species can help to understand TE variability and thalidomide’s teratogenic properties; hence it must be further investigated. http://dx.doi.org/10.1016/j.reprotox.2017.06.062 High sucrose low copper diet in diabetic and non-diabetic pregnant rats induces long-term changes in the offspring epigenome manifested by DNA hypo-methylation of critical developmental genes A. Ornoy ∗ , L. Weinstein-Fudim, M. Szyf, Z. Ergaz Laboratory of Teratology, Department of Medical Neurobiology, Hebrew University Hadassah Medical School, Jerusalem, Israel
a
INAGEMP – Instituto Nacional de Genética Médica Populacional, Porto Alegre, Brazil b Post-Graduate Program in Genetics and Molecular Biology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil c Laboratory of Medical Genetics and Evolution, Genetics Department, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Introduction: Thalidomide embryopathy (TE) phenotype is different across species, although a wide range of vertebrates are affected by its teratogenicity. One of the main mysteries in thalidomide history is why mice and rats present a higher resistance towards its teratogenic properties, when compared to other animals. The aim of this work is to investigate gene expression changes in components of thalidomide main affected pathways, Cereblon complex and angiogenesis, in more and less sensitive species. Here, we present our preliminary results. Methods: Transcriptome assays were accessed through Gene Expression Omnibus (GEO) database. We selected one Mus musculus Affymetrix transcriptome (GSE61306), performed in murine embryonic stem cells (mESC). Gene expression of mESC exposed to saline solution or thalidomide was accessed 24, 48 and 72 h after exposure. All data was evaluated in RStudio (v.1.0.136) through oligo package. Normalization of gene expression was performed by robust multi-array averaging (RMA). Differences between control and exposed groups of Crbn, Ddb1, Cul4a, Rbx1, Ikzf1, Ikzf3 (of the Cereblon complex), Nos3 and Vegfa (angiogenesis components) in the three mentioned periods were compared through t-Test or Mann–Whitney, according to the data distribution. Results: Preliminary results show that, regarding the genes evaluated, Cul4a was the only one statistically affected by
Introduction: We have previously shown that culture of 10 day old rat embryos in hyperglycemic, hyper-ketonemic culture medium induces a high rate of congenital anomalies and changes in SOD and Catalase gene expression. Moreover, the term placentae of Cohen diabetic CDs rats exhibited a variety of epigenetic changes, manifested by a reduction in total DNA methylation and, more specifically, in candidate diabetes genes and in promoters of various genes involved in embryonic development such as neuropeptide Y receptor, leptin and adiponectin. Methods: Studies were carried out on the offspring of CDs diabetic rats fed a high sucrose low copper diet (HSD) and of nondiabetic CDs rats fed regular diet (RD) compared to offspring of non-diabetic Sabra strain rats fed similar diets. We assessed in the offspring’ livers on days 3, 6, 13, 20 and 30 the redox status using biochemical and immunohistochemical methods, and DNA methylation by immunoprecipitation and pyrosequencing. Following delivery, all dams were fed RD. Results: HSD induced in the liver increased oxidative stress manifested by increased lipid peroxidation, increased SOD activity and reduced CAT activity. Immunohistochemistry showed elevated nitrotyrosine, increased proliferation and apoptosis and increased HIF␣-a marker of hypoxia. These changes were more prominent in the CDs rats compared to Sabra and were evident only in the first post-natal week, except HIF1␣, that was significantly increased in the CDs fed HSD for a longer period. Total DNA methylation was decreased by 25% in the livers of the CDs fed HSD compared to CDs fed RD. These DNA methylation changes were evident throughout the first postnatal month. The genes mostly affected were related to general developmental processes, metabolic and cardiovascular diseases.
