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

morphology

Ivana Bošnjak, PhDLaboratory for Biology and Molecular GeneticsFaculty of Food Technology and BiotechnologyUniversity of Zagreb, Croatia

2nd International Symposium “VERA JOHANIDES”Zagreb, Croatia, May 10-11 2013

Bisphenol A (BPA)

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….….

IUPAC: 4,4'-(propane-2,2-diyl)diphenol

Resin identification code

Toxicity of BPA to aquatic biota• Reported EC50 and LC50 values: 1 – 10 mg/L [4.4 –

43.8 µM]

Flint et al., 2012 Journal of Environmental Management 104: 19-34

“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…

AIMs:

Paracentrotus lividus: 2-cell pluteus

1. Real time quantitative PCR (qPCR) measurement:

2. Insight of ultramorphological changes of treated embryos by:A. transmission electron microscopy (TEM)B. scanning electron microscopy (SEM)

BPA exposure

Cellular mechanism Protein Target gene expressions

A. multixenobitoic resistance (MXR) mechanismchemical defensome

P-glycoprotein/P-gp abcb1

B. endocrine disruption orphan Steroid Hormone Receptor/SHR2 shr2

C. cell-cycle regulation Cyclin BCdk (Cyclin-dependent kinase)

cyccdk

ABC (ATP-Binding Cassette) transportersuse ATP for active transport of toxic compound across cell membrane (“efflux transporters”)P-glycoprotein/P-gp ABCB1 member of ABCB subfamily (abcb1 gene)

! high expression throughout sea urchin embryo development [Hamdoun et al., 2004; Shipp et al., 2012]

MXR mechanism Protection from vast variety of natural and anthropogenic toxic compounds present in aquatic environment. [Kurelec, 1992]

FIRST LINE OF

DEFENSE

0' 25' 30'

Fertilization

egg

Embryonic development (T = 16 °C)Time Post

Fertilization (PF):

Active MXR mechanism [Hamdoun et al.,

2004]

ADD BPA

1h35'

spermFertilization

envelop

96h

EXPERIMENTAL APPROACH:

Sample collection for:qPCR

TEM & SEM

100 nM [22.8 µg/L]

4 µM [910 µg/L]

100 nM [22.8 µg/L]

4 µM [910 µg/L]

EC50 = 2.5 µM [570 µg/L]

Toxicity of BPA on first cell division:

means ± SDs of 5 batches of embryos

qPCR results:

abcb1 (P-glycoprotein)

cyc (Cyclin B) shr2 (Shr2)

cdk (Cdk)

**

**

three independent RNA isolations of each egg culture normalised to ubiquitin mRNA bars represent means ±SD; * p < 0.05

5.3-fold

6.2-fold

2.7-fold 2.2 -

fold

1h 35’96h

Endocrine disruption

Cell cycle regulation

MXR mechanism

4 μM BPA2-cell stage

control

4 μM BPApluteus stage

control

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

Acknowledgements:

1 Ivona Mladineo, PhD

2Maria Ina Arnone, PhD2Rossella Annunziata , PhD

2 Marco Borra, PhD

2 Giovanna Benvenuto, PhD2 Davide Ciaramiello, facility

technician

1Institute of Oceanography and Fisheries, Split, Croatia

2Stazione Zoologica Anton Dohrn, Napoli, Italy

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: Canada2009: USA2011: EuropeEmbargo for BPA in baby bottles!

BPA is toxic!