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Alpha-Fe2O3 elicits diameter-dependent effects during exposure to an in vitro model of the human placenta

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Abstract

Iron oxide nanoparticles offer unique possibilities due to the change in their physico-chemical parameters when synthesized on the nanoscale (10−9 m) compared to their bulk forms. While novel uses exist for these materials when synthesized as nanoparticles, their unintended effects on the human body and specifically during pregnancy remain ill defined. In this study, an iron oxide nanoparticle, α-Fe2O3, was employed and the potential toxicity due to exposure was assessed in the widely used model human placental cell line BeWo b30. These cells were grown as epithelia, and subsequently assessed for their epithelial integrity, reactive oxygen species production and cellular viability, ultrastructural and morphological disruption, and genotoxicity as a result of exposure to α-Fe2O3 nanoparticles. Transepithelial electrical resistance indicated that exposure to the large (50 and 78 nm), but not small (15 nm) diameter particles of α-Fe2O3 nanomaterial resulted in leakiness of the epithelium. Exposure to the large diameters of 50 and 78 nm resulted in increases in cell death and reactive oxygen species. Disruption of junctional integrity as monitored by immunolocalization of the tight junction protein ZO-1 was found to occur as a consequence of exposure to large diameter NPs. It was found that there was reduction in the number of microvilli responsible for increased surface area for nutrient absorption after exposing the epithelia to large diameter NPs. Finally, genotoxicity as assessed by DNA microarray and confirmed by QPCR indicated that the large diameter particles (78 nm) induce apoptosis in these cells. These data indicate that large (50 and 78 nm), but not small (15 nm) α-Fe2O3 nanoparticles disrupt the barrier function of this epithelium as assessed by in vitro analysis.

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Abbreviations

BSA:

Bovine serum albumin

DAPI:

4′,6-Diamidino-2-phenylindole

DLS:

Dynamic light scattering

ICB:

Intracellular buffer

mRNA:

Messenger RNA

NP(s):

Nanoparticle

PBS:

Phosphate-buffered saline

ROS:

Reactive oxygen species

SEM:

Scanning electron microscopy

TEER:

Transepithelial electrical resistance

TEM:

Transmission electron microscopy

ZO-1:

Zonula Occludens-1

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Acknowledgments

The authors are indebted to Professor Erik Rytting at UTMB for providing the BeWo b30 cell line employed during this study. Professor Kaushal Rege and Dr. Thrimoorthy Potta are greatly thanked for their helpful discussions of this research. The authors thank Dr. Scott Bingham for his unique willingness to provide assistance in the DNA Core Facility at Arizona State University. The authors thank Mr. David Lowry for his patience training JJF on the scanning scope. All imaging data was procured in the W.M. Keck Bioimaging Facility at Arizona State University. JJF is supported in part by the McKee Award funded by the Delta Sigma Phi Foundation, and the Dr. and Mrs. John Maher Scholarship. This study was partially supported by the US Environmental Protection Agency Science to Achieve Results Program grant RD-83385601 and National Science Foundation Grant CBET-1235166.

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The authors declare no competing interests.

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Authors and Affiliations

  1. School of Life Sciences, Cellular and Molecular Biosciences, Arizona State University, Tempe, AZ, 85287-4501, USA

    James J. Faust & David G. Capco

  2. Department of Civil and Environmental Engineering, New Jersey Institute of Technology, Newark, NJ, 07102, USA

    Wen Zhang

  3. School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA, 30332, USA

    Yongsheng Chen

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  1. James J. Faust
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  4. David G. Capco
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Correspondence to Yongsheng Chen or David G. Capco.

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Supplemental Figure 1

Raw TEER values before percent normalization for epithelia exposed to NPs at concentrations of 100 μg/mL and 10 μg/mL. The histograms indicate that exposure to large diameter NPs result in disruption of TEER. (A) The histogram illustrates the change in TEER after application of different diameters of α-Fe2O3 at a concentration of 100 μg/mL. Both 50- and 78 nm NP treated epithelia follow the same trend, whereas the 15-nm diameter exposure followed the trend of the untreated specimens. (B) Exposure to 10 μg/mL for all α-Fe2O3 diameters tested results in no change compared to the untreated specimens. As indicated in the Methods section, TEER levels off at its maximum value of 40 Ωcm2 3 days after seeding BeWo cells. The NPs were applied after this 3 day culture period which is denoted as t = 0 in the graphs. All experiments were conducted at least three independent times where n = 3 (JPEG 70 kb)

High resolution image (TIFF 8084 kb)

Supplemental Figure 2

Tight junctions, as measured by ZO-1 immunofluorescence, are unperturbed after exposure to 15 nm α-Fe2O3 NPs at a 100 μg/mL concentration at the 1 day time point. After exposure to 15 nm α-Fe2O3 NPs the typical contiguous, honeycomb appearance of ZO-1 is seen in these specimens, albeit with modest discontinuity. (JPEG 86 kb)

High resolution image (TIFF 2102 kb)

Supplemental Figure 3

Morphological analysis of microvilli in 15 nm-treated specimens indicates no change in the number and structure of the microvilli. After exposure to 15 nm NPs the microvilli remain erect and appear to contain a similar number of microvilli compared to controls. (JPEG 32 kb)

High resolution image (TIFF 641 kb)

Supplemental Table 1

Detailed physico-chemical parameters of the NPs in medium indicates a change in ζ-potential as well as a slight degree of agglomeration over the course of 24 h. In ddH2O the ζ-potential of the nanomaterial remains stable and positive. However, after incubation in electrolyte-containing medium with serum there is a rapid shift to a negative ζ-potential. Further, the peak diameter grows slightly as a function of time. (JPEG 94 kb)

High resolution image (TIFF 668 kb)

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Faust, J.J., Zhang, W., Chen, Y. et al. Alpha-Fe2O3 elicits diameter-dependent effects during exposure to an in vitro model of the human placenta. Cell Biol Toxicol 30, 31–53 (2014). https://doi.org/10.1007/s10565-013-9267-9

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