Article | Published:

Nanoparticle-induced neuronal toxicity across placental barriers is mediated by autophagy and dependent on astrocytes


The potential for maternal nanoparticle (NP) exposures to cause developmental toxicity in the fetus without the direct passage of NPs has previously been shown, but the mechanism remained elusive. We now demonstrate that exposure of cobalt and chromium NPs to BeWo cell barriers, an in vitro model of the human placenta, triggers impairment of the autophagic flux and release of interleukin-6. This contributes to the altered differentiation of human neural progenitor cells and DNA damage in the derived neurons and astrocytes. Crucially, neuronal DNA damage is mediated by astrocytes. Inhibiting the autophagic degradation in the BeWo barrier by overexpression of the dominant-negative human ATG4BC74A significantly reduces the levels of DNA damage in astrocytes. In vivo, indirect NP toxicity in mice results in neurodevelopmental abnormalities with reactive astrogliosis and increased DNA damage in the fetal hippocampus. Our results demonstrate the potential importance of autophagy to elicit NP toxicity and the risk of indirect developmental neurotoxicity after maternal NP exposure.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Anselmo, A. C. & Mitragotri, S. Nanoparticles in the clinic. Bioeng. Transl. Med. 1, 10–29 (2016).

  2. 2.

    Saei, A. A. et al. Nanoparticle surface functionality dictates cellular and systemic toxicity. Chem. Mater. 29, 6578–6595 (2017).

  3. 3.

    Thubagere, A. & Reinhard, B. M. Nanoparticle-induced apoptosis propagates through hydrogen-peroxide-mediated bystander killing: insights from a human intestinal epithelium in vitro model. ACS Nano 4, 3611–3622 (2010).

  4. 4.

    Bhabra, G. et al. Nanoparticles can cause DNA damage across a cellular barrier. Nat. Nanotech. 4, 876–883 (2009).

  5. 5.

    Sood, A. et al. Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness. Nat. Nanotech. 6, 824–833 (2011).

  6. 6.

    Grandjean, P. & Landrigan, P. J. Neurobehavioural effects of developmental toxicity. Lancet Neurol. 13, 330–338 (2014).

  7. 7.

    Shimizu, M. et al. Maternal exposure to nanoparticulate titanium dioxide during the prenatal period alters gene expression related to brain development in the mouse. Part. Fibre Toxicol. 6, 20 (2009).

  8. 8.

    Hougaard, K. S. et al. Effects of prenatal exposure to surface-coated nanosized titanium dioxide (UV-Titan). A study in mice. Part. Fibre Toxicol. 7, 16 (2010).

  9. 9.

    Mohammadipour, A. et al. Maternal exposure to titanium dioxide nanoparticles during pregnancy; impaired memory and decreased hippocampal cell proliferation in rat offspring. Environ. Toxicol. Pharmacol. 37, 617–625 (2014).

  10. 10.

    Blum, J. L., Xiong, J. Q., Hoffman, C. & Zelokoff, J. T. Cadium associated with inhaled cadium oxide nanoparticles impacts fetal and neonatal development and growth. Toxicol. Sci. 126, 478–486 (2012).

  11. 11.

    Goeden, N. et al. Maternal inflammation disrupts fetal neurodevelopment via increased placental output of serotonin to the fetal brain. J. Neurosci. 36, 6041–6049 (2016).

  12. 12.

    Li, H., van Ravenzwaay, B., Rietjens, I. M. & Louisse, J. Assessment of an in vitro transport model using BeWo b30 cells to predict placental transfer of compounds. Arch. Toxicol. 87, 1661–1669 (2013).

  13. 13.

    Bode, C. J. et al. In vitro models for studying trophoblast transcellular transport. Methods Mol. Med. 122, 225–239 (2006).

  14. 14.

    Polyzois, I., Nikolopoulos, D., Michos, I., Patsouris, E. & Theocharis, S. Local and systemic toxicity of nanoscale debris particles in total hip arthroplasty. J. Appl. Toxicol. 32, 255–269 (2012).

  15. 15.

    Papageorgiou, I. et al. The effect of nano- and micron-sized particles of cobalt–chromium alloy on human fibroblasts in vitro. Biomaterials 28, 2946–2958 (2007).

  16. 16.

    Crighton, D. et al. DRAM, a p53-induced modulator of autophagy, is critical for apoptosis. Cell 126, 121–134 (2006).

  17. 17.

    Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1–222 (2016).

  18. 18.

    Ma, X. et al. Gold nanoparticles induce autophagosome accumulation through size-dependent nanoparticle uptake and lysosome impairment. ACS Nano 5, 8629–8639 (2011).

  19. 19.

    Svendsen, C. N. et al. A new method for the rapid and long term growth of human neural precursor cells. J. Neurosci. Methods 85, 141–152 (1998).

  20. 20.

    Sofroniew, M. V. Multiple roles for astrocytes as effectors of cytokines and inflammatory mediators. Neuroscientist 20, 160–172 (2014).

  21. 21.

    Dickey, J. S. et al. Intercellular communication of cellular stress monitored by γ-H2AX induction. Carcinogenesis 30, 1686–1695 (2009).

  22. 22.

    Paull, T. T. et al. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886–895 (2000).

  23. 23.

    Basak, R. & Bandyopadhyay, R. Encapsulation of hydrophobic drugs in Pluronic F127 micelles: effects of drug hydrophobicity, solution temperature, and pH. Langmuir 29, 4350–4356 (2013).

  24. 24.

    Chen, Y. et al. Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism. J. Neurochem. 77, 1601–1610 (2001).

  25. 25.

    Barnabe-Heider, F. et al. Evidence that embryonic neurons regulate the onset of cortical gliogenesis via cardiotrophin-1. Neuron 48, 253–265 (2005).

  26. 26.

    Pal, R., Mamidi, M. K., Das, A. K. & Bhonde, R. Human embryonic stem cell proliferation and differentiation as parameters to evaluate developmental toxicity. J. Cell Physiol. 226, 1583–1595 (2011).

  27. 27.

    Pevny, L. H., Sockanathan, S., Placzek, M. & Lovell-Badge, R. A role for SOX1 in neural determination. Development 125, 1967–1978 (1998).

  28. 28.

    Dieriks, B., de Vos, W. H., Derradji, H., Baatout, S. & van Oostveldt, P. Medium-mediated DNA repair response after ionizing radiation is correlated with the increase of specific cytokines in human fibroblasts. Mutat. Res. 687, 40–48 (2010).

  29. 29.

    Fujita, N. et al. An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol. Biol. Cell 19, 4651–4659 (2008).

  30. 30.

    Betin, V. M., Singleton, B. K., Parsons, S. F., Anstee, D. J. & Lane, J. D. Autophagy facilitates organelle clearance during differentiation of human erythroblasts: evidence for a role for ATG4 paralogs during autophagosome maturation. Autophagy 9, 881–893 (2013).

  31. 31.

    Maycotte, P., Jones, K. L., Goodall, M. L., Thorburn, J. & Thorburn, A. Autophagy supports breast cancer stem cell maintenance by regulating IL6 secretion. Mol. Cancer Res. 13, 651–658 (2015).

  32. 32.

    Narita, M. et al. Spatial coupling of mTOR and autophagy augments secretory phenotypes. Science 332, 966–970 (2011).

  33. 33.

    Chen, Z. H. et al. Autophagy is essential for ultrafine particle-induced inflammation and mucus hyperproduction in airway epithelium. Autophagy 12, 297–311 (2016).

  34. 34.

    Hunter, C. A. & Jones, S. A. IL-6 as a keystone cytokine in health and disease. Nat. Immunol. 16, 448–457 (2015).

  35. 35.

    Mauer, J., Denson, J. L. & Bruning, J. C. Versatile functions for IL-6 in metabolism and cancer. Trends Immunol. 36, 92–101 (2015).

  36. 36.

    Campbell, I. L. et al. Neurologic disease induced in transgenic mice by cerebral overexpression of interleukin 6. Proc. Natl Acad. Sci. USA 90, 10061–10065 (1993).

  37. 37.

    Shinozaki, Y. et al. Transformation of astrocytes to a neuroprotective phenotype by microglia via P2Y1 receptor downregulation. Cell Rep. 19, 1151–1164 (2017).

  38. 38.

    Haroon, F. et al. Gp130-dependent astrocytic survival is critical for the control of autoimmune central nervous system inflammation. J. Immunol. 186, 6521–6531 (2011).

  39. 39.

    Drogemuller, K. et al. Astrocyte gp130 expression is critical for the control of Toxoplasma encephalitis. J. Immunol. 181, 2683–2693 (2008).

  40. 40.

    Rothaug, M., Becker-Pauly, C. & Rose-John, S. The role of interleukin-6 signaling in nervous tissue. Biochim. Biophys. Acta 1863, 1218–1227 (2016).

  41. 41.

    Simmons, D. G. et al. Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth. Development 135, 2083–2091 (2008).

  42. 42.

    Hennessy, E., Griffin, E. W. & Cunningham, C. Astrocytes are primed by chronic neurodegeneration to produce exaggerated chemokine and cell infiltration responses to acute stimulation with the cytokines IL-1β and TNF-α. J. Neurosci. 35, 8411–8422 (2015).

  43. 43.

    Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487 (2017).

  44. 44.

    Zamanian, J. L. et al. Genomic analysis of reactive astrogliosis. J. Neurosci. 32, 6391–6410 (2012).

  45. 45.

    Broad, K. D. & Keverne, E. B. Placental protection of the fetal brain during short-term food deprivation. Proc. Natl Acad. Sci. USA 108, 15237–15241 (2011).

  46. 46.

    Avagliano, L. et al. Autophagy in placentas from acidotic newborns: an immunohistochemical study of LC3 expression. Placenta 34, 1091–1094 (2013).

  47. 47.

    Stern, S. T., Adiseshaiah, P. P. & Crist, R. M. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part. Fibre Toxicol. 9, 20 (2012).

  48. 48.

    Sun, W. et al. Glutamate-dependent neuroglial calcium signaling differs between young and adult brain. Science 339, 197–200 (2013).

  49. 49.

    Ostenfeld, T. & Svendsen, C. N. Recent advances in stem cell neurobiology. Adv. Tech. Stand. Neurosurg. 28, 3–89 (2003).

  50. 50.

    Danson, C. M., Pocha, S. M., Bloomberg, G. B. & Cory, G. O. Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity. J. Cell Sci. 120, 4144–4154 (2007).

Download references


S.J.H. was supported by a fellowship from the Bristol Orthopaedic Trust. Work in the Caldwell lab is supported by Parkinson’s UK, James Tudor Foundation and EPSRC. We thank A. Schwartz (Washington University in St Louis) for permission to use the BeWo b30 cells, G. Cory (University of Exeter) for the modified pSEW sin vector and A. Rosser (Cardiff University) for providing the human fetal tissue for this work. The Cardiff Fetal Tissue Bank is funded by the MRC, NISCHR and Cardiff University. We also thank A. Blom for advice, A. Herman, S. Chappell, the University of Bristol Faculty of Biomedical Sciences Flow Cytometry Facility and I. T. Chang (School of Metallurgy and Materials, University of Birmingham).

Author information

S.J.H., C.P.C. and M.A.C. conceived and designed the experiments, and C.P.C. and M.A.C. directed the work. S.J.H. completed the experiments with the help of L.A.C., O.C.-L., P.S., N.J.-M., S.F.M., C.E.G. and A.S., and also completed the data analysis with the help of M.A.C. J.D.L. oversaw the autophagy experiments and the lentiviral shRNA experiments, and M.S. provided advice on the BeWo barriers. S.K. performed the in vivo injections and the analysis was completed by A.B., N.T.B. and A.M.M. S.J.H., C.P.C. and M.A.C. wrote the paper and all the authors commented on it and agreed the final version.

Competing interests

The authors declare no competing interests.

Correspondence to Maeve A. Caldwell.

Supplementary information

Supplementary Information

Supplementary Figures 1–22, Supplementary methods, Supplementary Tables 1–3, Supplementary references.

Life Sciences Reporting Summary

Flow Cytometry Checklist

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading

Fig. 1: NPs alter the autophagic flux in the upper layers of the BeWo barrier.
Fig. 2: Astrocytes in mixed NPC culture undergo morphological changes after indirect exposure to NPs.
Fig. 3: NPs increase the number of γ-H2AX foci in mixed astrocyte and neuronal cultures.
Fig. 4: Mechanism of NP-induced indirect toxicity is dependent on the presence of astrocytes and is triggered by autophagy in the BeWo barrier.
Fig. 5: GFAP and γ-H2AX levels are increased in the hippocampus of neonates after maternal exposure to CoCr NPs at E12.5.