Toxicity of bisphenol A (BPA) on

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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
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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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Resin identification code
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Toxicity of BPA to aquatic biota

• Reported EC50 and LC50 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 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
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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)

Cellular mechanism Protein Target gene expressions
A. multixenobitoic resistance (MXR) mechanism chemical defensome P-glycoprotein/P-gp abcb1
B. endocrine disruption orphan Steroid Hormone Receptor/SHR2 shr2
C. cell-cycle regulation Cyclin B Cdk (Cyclin-dependent kinase) cyc cdk
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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 (abcb1 gene)
• ! high expression throughout sea urchin embryo
  development
• Hamdoun et al., 2004 Shipp et al., 2012

FIRST LINE OF DEFENSE
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EXPERIMENTAL APPROACH
100 nM 22.8 µg/L 4 µM 910 µg/L
96h
sperm
Fertilization envelop
Sample collection for qPCR TEM SEM
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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

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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 lt 0.05

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• 4 µM BPA
• 2-cell stage

control
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• 4 µM BPA
• pluteus stage

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