Toxicity of bisphenol A BPA on urchin embryo

Toxicity of bisphenol A (BPA) on urchin embryo gene expression and morphology Ivana Bošnjak, Ph. D Laboratory for Biology and Molecular Genetics Faculty of Food Technology and Biotechnology University of Zagreb, Croatia 2 nd International Symposium “VERA JOHANIDES” Zagreb, Croatia, May 10 -11 2013

Bisphenol A (BPA) IUPAC: 4, 4'-(propane-2, 2 -diyl)diphenol 1891: Chemical synthesis of BPA in the laboratory (A. Dianin, Russia) 1953: Start of BPA global production polycarbonate polymers and epoxy resins 2011: 5. 5 million metric tons per year (Greiner et al. , 2007) 2015: 7 million metric tons per year (China Chemical Industry News 2005) BPA plastic: • Cheap, useful, tough, resistible, transparent…. ….

Resin identification code

Toxicity of BPA to aquatic biota • Reported EC 50 and LC 50 values: 1 – 10 mg/L [4. 4 – 43. 8 µM] “moderately toxic” and “toxic” to aquatic biota [European Commission & United States Environmental Protection Agency (US EPA)] ! Harmful even at environmentally relevant concentrations: 12 µg/L or lower [52. 6 n. M or lower] BPA toxicity studies: endocrine-related measurement endpoints - e. g. enlarged sex glands, oviduct deformities, increased fecundity, additional female organs, development arrest… Flint et al. , 2012 Journal of Environmental Management 104: 19 -34

BPA exposure AIMs: Paracentrotus lividus: 2 -cell pluteus 1. Real time quantitative PCR (q. PCR) measurement: 2. Cellular mechanism Protein A. multixenobitoic resistance (MXR) mechanism chemical defensome P-glycoprotein/P-gp Target gene expressions abcb 1 B. endocrine disruption orphan Steroid Hormone Receptor/SHR 2 shr 2 C. cell-cycle regulation Cyclin B Cdk (Cyclin-dependent kinase) cyc cdk Insight of ultramorphological changes of treated embryos by: A. transmission electron microscopy (TEM) B. scanning electron microscopy (SEM)

MXR mechanism Protection from vast variety of natural and anthropogenic toxic compounds present in aquatic environment. [Kurelec, 1992] ABC (ATP-Binding Cassette) transporters v use ATP for active transport of toxic compound across cell membrane (“efflux transporters”) FIRST v P-glycoprotein/P-gp LINE OF ABCB 1 member of ABCB subfamily (abcb 1 gene) DEFENSE ! high expression throughout sea urchin embryo development [Hamdoun et al. , 2004; Shipp et al. , 2012]

EXPERIMENTAL APPROACH: Active MXR mechanism [Hamdoun et al. , 2004] 100 n. M [22. 8 μg/L] ADD BPA 4 μM [910 μg/L] Embryonic development (T = 16 °C) Time Post Fertilization (PF): 0' 25' 30' 96 h 1 h 35' Fertilization sperm egg Fertilization envelop Sample collection for: q. PCR TEM & SEM

Toxicity of BPA on first cell division: EC 50 = 2. 5 μM [570 μg/L] 100 n. M [22. 8 μg/L] 4 μM [910 μg/L] means ± SDs of 5 batches of embryos

q. PCR results: MXR mechanism abcb 1 (Pglycoprotein) Endocrine disruption shr 2 (Shr 2) cyc (Cyclin B) Cell cycle cdk (Cdk) * 5. 3 -fold * 6. 2 -fold regulation * 2. 7 -fold 2. 2 -fold * 1 h 35’ three independent RNA isolations of each egg culture normalised to ubiquitin m. RNA bars represent means ±SD; * p < 0. 05 96 h

v 4 μM BPA v 2 -cell stage control

v 4 μM BPA v pluteus stage control

Conclusions: v EC 50 = 2. 5 μM BPA [570 µg/L] cell-cycle arrest or delay v Target genes expression (q. PCR): 100 n. M & 4 μM BPA significant upregulation of abcb 1 gene (P-gp expression) = involvement of MXR mechanism! 4 μM BPA = upregulation of other target genes: shr 2, cyc and cdk v SEM & TEM results: Endocrine disruption Cell cycle regulation higher sublethal concentration of BPA (4 μM) induces disorder in karyokinesis and developmental retardation

Acknowledgements: 1 Ivona Mladineo, Ph. D 2 Maria Ina Arnone, Ph. D 2 Rossella Annunziata , Ph. D 2 Marco Borra, Ph. D 2 Giovanna Benvenuto, Ph. D 2 Davide Ciaramiello, facility technician 1 Institute of Oceanography and Fisheries, Split, Croatia 2 Stazione Zoologica Anton Dohrn, Napoli, Italy

BPA is toxic! Endocrine disruptor: • Cause hormone chaos • Metabolism disorder, immunity disorder, affects growth and development during childhood, affects behavior, nerve and cardiovascular system disorder, cause breast cancer and prostate cancer, thyroid gland disorder…. . 2008: Canada 2009: USA 2011: Europe Embargo for BPA in baby bottles!