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Thomas C. Long,¹ Julianne Tajuba,¹ Preethi Sama,¹ Navid Saleh,² Carol Swartz,¹ Joel Parker,³ Susan Hester,⁴ Gregory V. Lowry,² and Bellina Veronesi⁴

¹Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina, USA; ²Department of Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA; ³Constella Inc., Research Triangle Park, North Carolina, USA; ⁴National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA

Abstract
Background: Titanium dioxide is a widely used nanomaterial whose photo-reactivity suggests that it could damage biological targets (e.g., brain) through oxidative stress (OS) .

Objectives: Brain cultures of immortalized mouse microglia (BV2) , rat dopaminergic (DA) neurons (N27) , and primary cultures of embryonic rat striatum, were exposed to Degussa P25, a commercially available TiO₂ nanomaterial. Physical properties of P25 were measured under conditions that paralleled biological measures.

Findings: P25 rapidly aggregated in physiological buffer (800–1,900 nm ; 25°C) and exposure media (~ 330 nm ; 37°C) , and maintained a negative zeta potential in both buffer (–12.2 ± 1.6 mV) and media (–9.1 ± 1.2 mV) . BV2 microglia exposed to P25 (2.5–120 ppm) responded with an immediate and prolonged release of reactive oxygen species (ROS) . Hoechst nuclear stain was reduced after 24-hr (? 100 ppm) and 48-hr (? 2.5 ppm) exposure. Microarray analysis on P25-exposed BV2 microglia indicated up-regulation of inflammatory, apoptotic, and cell cycling pathways and down-regulation of energy metabolism. P25 (2.5–120 ppm) stimulated increases of intracellular ATP and caspase 3/7 activity in isolated N27 neurons (24–48 hr) but did not produce cytotoxicity after 72-hr exposure. Primary cultures of rat striatum exposed to P25 (5 ppm) showed a reduction of immunohistochemically stained neurons and microscopic evidence of neuronal apoptosis after 6-hr exposure. These findings indicate that P25 stimulates ROS in BV2 microglia and is nontoxic to isolated N27 neurons. However, P25 rapidly damages neurons at low concentrations in complex brain cultures, plausibly though microglial generated ROS.

Key words: BV2, environmental nanotoxicity, neurotoxicity, oxidative stress, P25, titanium dioxide. Environ Health Perspect 115:1631–1637 (2007) . doi:10.1289/ehp.10216 available via http://dx.doi.org/ [Online 3 August 2007]

Address correspondence to B. Veronesi, U.S. EPA, NHEERL, NTD B105-06, 109 T.W. Alexander Dr., Research Triangle Park, NC 27711 USA. Telephone: (919) 541-5780. Fax: (919) 541-4849. E-mail: veronesi.bellina@epa.gov

The authors acknowledge the kind gift of P25 from Degussa Corp. (Frankfurt/Main, Germany) and also thank J. Hong of the National Institute of Environmental Health Sciences, Research Triangle Park, NC, for his gift of the BV2 and N27 cell lines. The authors also acknowledge the expert preparation of electron microscopy samples by the Microscopy Services Laboratory, University of North Carolina, Chapel Hill, North Carolina.

The technical support of T.C. Long was supported by U.S. EPA Professional Services Contract X3C247. T.L. was supported under U.S. EPA service contract X3427.

This document has been reviewed by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute the endorsement of recommendation for use.

The authors declare they have no competing financial interests.

Received 2 March 2007 ; accepted 3 August 2007.

Correction

Authorship of this article has been modified from the orignal article published online.