Sensitive neurotoxicity assessment of bisphenol A using double immunocytochemistry of DCX and MAP2

다국어 초록 (Multilingual Abstract)

Bisphenol A (BPA) is an environmental toxin widely used in manufacturing industries. Studies conducted on the neurotoxicity of BPA demonstrated that at excessive, high concentrations (≥ 200 µM) adverse responses occurred which were not detectable using traditional toxicity tests at lower chemical quantities than 200 µM. Thus, a method capable of effectively detecting neurotoxicity at low concentrations (≤ 100 µM) was devised. Bisphenol A-mediated neurotoxicity was examined in primary cultured neurons using various methods, including Western blot, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity and reactive oxygen species assays. These methods confirmed BPA-induced toxicity at 200 μM, but no marked effect was observed at concentrations below 200 μM. However, when immunocytochemistry (ICC) was performed using a co-immunofluorescence assay of doublecortin (DCX) and microtubule-associated protein 2 (MAP2), BPA adversely affected neuronal maturation in neural progenitor cells at concentrations as low as 100 µM, at which the three traditional methods failed to detect any neurotoxic effect. Our DCX/MAP2 ICC findings indicate that low concentrations of BPA are toxic to developing neurons, and suggest that the devised double ICC technique might provide an effective means of assessing neurotoxic effects of environmental toxins at low concentrations.

참고문헌 (Reference)

1 Liao CY, "Widespread occurrence of bisphenol A in paper and paper products : implications for human exposure" 45 : 9372-9379, 2011

2 Calafat AM, "Urinary concentrations of bisphenol A and 4-nonylphenol in a human reference population" 113 : 391-395, 2005

3 Becher A, "The synaptophysin-synaptobrevin complex : a hallmark of synaptic vesicle maturation" 19 : 1922-1931, 1999

4 Zhang M, "The influence of Schisandrin B on a model of Alzheimer’s disease using beta-amyloid protein A beta(1-42)-mediated damage in SH-SY5Y neuronal cell line and underlying mechanisms" 80 : 1199-1205, 2017

5 Flynn KC, "The cytoskeleton and neurite initiation" 3 : 86-109, 2013

6 Sarnat HB, "Synaptophysin immunoreactivity in the human hippocampus and neocortex from 6 to 41 weeks of gestation" 69 : 234-245, 2010

7 Kim K, "Suppressive effects of bisphenol A on the proliferation of neural progenitor cells" 70 : 1288-1295, 2007

8 Lee S, "Signaling pathways of bisphenol A-induced apoptosis in hippocampal neuronal cells: role of calcium-induced reactive oxygen species, mitogen-activated protein kinases, and nuclear factor-kappaB" 86 : 2932-2942, 2008

9 Xu XH, "Sex-specific influence of exposure to bisphenol-A between adolescence and young adulthood on mouse behaviors" 61 : 565-573, 2011

10 Elsworth JD, "Prenatal exposure to bisphenol A impacts midbrain dopamine neurons and hippocampal spine synapses in non-human primates" 35 : 113-120, 2013

11 Kim K, "Potencies of bisphenol A on the neuronal differentiation and hippocampal neurogenesis" 72 : 1343-1351, 2009

12 Sarma SN, "Neurotoxicity of alkylated polycyclic aromatic compounds in human neuroblastoma cells" 80 : 285-300, 2017

13 Zhang J, "Molecular biomarkers for embryonic and adult neural stem cell and neurogenesis" 2015 : 727542-, 2015

14 Zhao C, "Mechanisms and functional implications of adult neurogenesis" 132 : 645-660, 2008

15 Kolker S, "Maturation-dependent neurotoxicity of 3-hydroxyglutaric and glutaric acids in vitro : a new pathophysiologic approach to glutaryl-CoA dehydrogenase deficiency" 47 : 495-503, 2000

16 Shin BS, "Maternal-fetal disposition of bisphenol A in pregnant Sprague-Dawley rats" 65 : 395-406, 2002

17 Wang Z, "Low-dose bisphenol A exposure : a seemingly instigating carcinogenic effect on breast cancer" 4 : 1600248-, 2017

18 Chen Z, "Long-term exposure to a ‘safe’ dose of bisphenol A reduced protein acetylation in adult rat testes" 7 : 40337-, 2017

19 Fernandes LS, "Land T-type calcium channel blockers protect against the inhibitory effects of mipafox on neurite outgrowth and plasticity-related proteins in SH-SY5Y cells" 80 : 1086-1097, 2017

20 Tiwari SK, "Inhibitory effects of bisphenol-A on neural stem cells proliferation and differentiation in the rat brain are dependent on Wnt/beta-catenin pathway" 52 : 1735-1757, 2015

21 Ooe H, "Induction of reactive oxygen species by bisphenol A and abrogation of bisphenol A-induced cell injury by DJ-1" 88 : 114-126, 2005

22 Wetherill YB, "In vitro molecular mechanisms of bisphenol A action" 24 : 178-198, 2007

23 Braun JM, "Impact of early-life bisphenol A exposure on behavior and executive function in children" 128 : 873-882, 2011

24 Jang YJ, "High dose bisphenol A impairs hippocampal neurogenesis in female mice across generations" 296 : 73-82, 2012

25 Wolstenholme JT, "Gestational exposure to low dose bisphenol A alters social behavior in juvenile mice" 6 : 25448-, 2011

26 Wang L, "Generation of integration-free neural progenitor cells from cells in human urine" 10 : 84-89, 2013

27 Calafat AM, "Exposure of the U.S. population to bisphenol A and 4-tertiaryoctylphenol: 2003-2004" 116 : 39-44, 2008

28 Rubin BS, "Evidence of altered brain sexual differentiation in mice exposed perinatally to low, environmentally relevant levels of bisphenol A" 147 : 3681-3691, 2006

29 Giordano G, "Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices : role of astrocytes and extracellular matrix proteins" 82 : 1792-1799, 2011

30 Yoot Mo Lee, "Estrogen receptor independent neurotoxic mechanism ofbisphenol A, an environmental estrogen" 대한수의학회 8 (8): 27-38, 2007

31 Palanza P, "Effects of developmental exposure to bisphenol A on brain and behavior in mice" 108 : 150-157, 2008

32 Kabuto H, "Effects of bisphenol A on the metabolisms of active oxygen species in mouse tissues" 93 : 31-35, 2003

33 Fujimura M, "Differing effects of toxicants (methylmercury, inorganic mercury, lead, amyloid beta, and rotenone) on cultured rat cerebrocortical neurons: differential expression of rho proteins associated with neurotoxicity" 126 : 506-514, 2012

34 Galloway T, "Daily bisphenol A excretion and associations with sex hormone concentrations : results from the InCHIANTI adult population study" 118 : 1603-1608, 2010

35 Rahman MS, "Bisphenol-A affects male fertility via fertility-related proteins in spermatozoa" 5 : 9169-, 2015

36 Mileva G, "Bisphenol-A : epigenetic reprogramming and effects on reproduction and behavior" 11 : 7537-7561, 2014

37 Hong SB, "Bisphenol A in relation to behavior and learning of school-age children" 54 : 890-899, 2013

38 Resnik DB, "Bisphenol A and risk management ethics" 29 : 182-189, 2015

39 Zalko D, "Biotransformations of bisphenol A in a mammalian model : answers and new questions raised by lowdose metabolic fate studies in pregnant CD1 mice" 111 : 309-319, 2003

40 Geens T, "Are potential sources for human exposure to bisphenol-A overlooked?" 214 : 339-347, 2011

41 Nowicki BA, "Adverse effects of bisphenol A(BPA)on the dopamine system in two distinct cell models and corpus striatum of the Sprague-Dawley rat" 79 : 912-924, 2016

42 Goncalves JT, "Adult neurogenesis in the hippocampus : from stem cells to behavior" 167 : 897-914, 2016

43 Agarwal S, "Activation of autophagic flux against xenoestrogen bisphenol-A-induced hippocampal neurodegeneration via AMP kinase(AMPK)/mammalian target of rapamycin(mTOR)pathways" 290 : 21163-21184, 2015