Toxicity of bisphenol A (BPA) on urchin embryo gene expression and morphology

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

2. 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….….

3. Resin identification code

4. Toxicity of BPA to aquatic biota • Reported EC50 and LC50values: 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 nM 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

5. BPA exposure AIMs: Paracentrotus lividus: 2-cell pluteus • Real time quantitative PCR (qPCR) measurement: • Insight of ultramorphological changes of treated embryos by: • transmission electron microscopy (TEM) • scanning electron microscopy (SEM)

6. MXR mechanism • Protection from vast variety of natural and anthropogenic toxic compounds present in aquatic environment. [Kurelec, 1992] • ABC (ATP-Binding Cassette)transporters • use ATP for active transport of toxic compound across cell membrane (“efflux transporters”) • P-glycoprotein/P-gp •  ABCB1 member of ABCB subfamily (abcb1gene) • ! high expression throughout sea urchin embryo development • [Hamdoun et al., 2004; Shipp et al., 2012] FIRST LINE OF DEFENSE

7. Active MXR mechanism [Hamdoun et al., 2004] ADD BPA Embryonic development (T = 16 °C) Time Post Fertilization (PF): 0' 25' 30' 1h35' Fertilization egg EXPERIMENTAL APPROACH: 100 nM [22.8 µg/L] 4 µM [910 µg/L] 96h sperm Fertilization envelop Sample collection for: qPCR TEM & SEM

8. Toxicity of BPA on first cell division: EC50 = 2.5 µM [570 µg/L] 100 nM [22.8 µg/L] 4 µM [910 µg/L] • means ± SDs of 5 batches of embryos

9. qPCR results: MXR mechanism 5.3-fold * abcb1 (P-glycoprotein) 6.2-fold * Endocrine disruption shr2 (Shr2) cyc (Cyclin B) Cell cycle regulation cdk (Cdk) 2.7-fold * 2.2 -fold * 1h 35’ 96h • three independent RNA isolations of each egg culture • normalised to ubiquitin mRNA • bars represent means ±SD; * p < 0.05

10. 4 μM BPA • 2-cell stage control

11. 4 μM BPA • pluteus stage control

12. Conclusions: • EC50 = 2.5 µM BPA [570 µg/L] • cell-cycle arrest or delay • Target genes expression (qPCR): • 100 nM& 4 µM BPA significant upregulation of abcb1 gene (P-gp expression) = involvement of MXR mechanism! • 4 µM BPA = upregulation of other target genes: shr2, cyc and cdk • SEM & TEM results: •  higher sublethal concentration of BPA (4 µM) induces disorder in karyokinesis and developmental retardation Endocrine disruption Cell cycle regulation

13. Acknowledgements: 1IvonaMladineo, PhD 2Maria Ina Arnone, PhD 2Rossella Annunziata , PhD 2 Marco Borra, PhD 2 Giovanna Benvenuto, PhD 2 DavideCiaramiello, facility technician 1Institute of Oceanography and Fisheries, Split, Croatia 2Stazione Zoologica Anton Dohrn, Napoli, Italy

15. 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!