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
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….….
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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:
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
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!