Spotlight Selective AHR Modulators Aryl hydrocarbon receptor (AHR) activation by xenobiotic compounds leads to nuclear translocation, dimerization with ARNT, and binding to dioxin responsive elements (DREs) to induce expression of metabolizing enzymes. However, AHR activation also leads to repression of anti-inflammatory signaling by a non-DRE-mediated mechanism. Thus, Murray, et al. [(2009) Mol. Pharmacol., DOI:10.1124/mol/ 109.061788, published online Nov 10] have proposed that selective AHR modulators (SAhRMs), which induce the immunosuppressive effects of the AHR in the absence of DRE-mediated enzyme induction, could be valuable therapeutic agents for inflammatory or autoimmune diseases. Murray et al. showed that the selective estrogen receptor (ER) modulator Way-169916 (Way) is also an AHR ligand. Even at high concentrations, Way stimulated
No Barrier to Nanotoxicity The rapid development of new forms of nanoparticles for a wide range of applications has led to growing concerns about their toxicity. A key unanswered question is whether epithelial barriers, such as exist in skin, lung, the gastrointestinal tract, and placenta, protect underlying tissues from nanoparticle-mediated damage. To address this question, Bahbra et al. [(2009) Nat. Nanotechnol. 4, 876] grew a confluent multilayer of BeWo cells in a transwell insert and investigated the ability of CoCr nanoparticles placed above the BeWo cells to inflict DNA damage on fibroblasts grown below the transwell. BeWo cells, a human trophoblast choriocarcinoma cell line, are used as an in vitro model epithelial barrier. Indirect 24 h exposure to CoCr nanoparticles through the BeWo barrier caused comparable levels of DNA damage to fibroblasts as direct exposure to nanoparticles in the fibroblast culture medium. When nanoparticles were placed in the transwells in the absence of BeWo cells, DNA damage to underlying fibroblasts was actually lower than when BeWo cells were present, suggesting that the BeWo cells contributed to the toxic mechanism. Microscopic and chemical analyses revealed that CoCr particles were taken up and partially degraded by the top layer of BeWo cells. There was no evidence of cell death, changes in permeability, or electrical resistance in the BeWo layer; however, a dose-dependent decrease in WST1 reduction by nanoparticle-exposed BeWo cells suggested mitochondrial toxicity. Blockers of cell-to-cell communications via connexin- or pannexin-dependent processes reduced indirect CoCr296
Vol. 23,
No. 2, •
CHEMICAL RESEARCH IN TOXICOLOGY
the expression of only insignificant levels of a CYP1A1 enhancer luciferase reporter in HepG2 cells. However, Way exhibited dose-dependent inhibition of the interleukin 1β (IL1β)-mediated acute phase response (APR) in Huh7 cells, as measured by the suppression of induction of three APR proteins. The effects of Way on the IL1β-mediated APR was not ER-dependent since it was not affected by an ER antagonist, and Huh7 cells do not express ERR or β. In contrast, combined siRNA knockdown of both AHR and ARNT blocked the inhibition of the IL1β-dependent APR by Way. The data support the intriguing hypothesis that SAhRMs, which selectively activate the physiologic functions of the AHR without inducing potentially toxic levels of xenobiotic metabolizing enzymes, may be of therapeutic value. • Carol A. Rouzer
mediated DNA damage of fibroblasts through the BeWo barrier. These findings suggested a role for purinergic (P2) receptors and ATP-dependent signaling, a hypothesis that was supported by the finding that direct exposure to ATP induced DNA damage in fibroblasts and that inhibitors of P2X signaling blocked indirect CoCr nanoparticle-mediated damage. ATP induced a spike in intracellular Ca2+ in BeWo cells, and inhibitors of Ca2+-dependent signaling also blocked indirect nanoparticle-mediated DNA damage in fibroblasts.
Reprinted with permission from Bahbra et al. [(2009) Nat. Nanotechnol. 4, 876]. Copyright 2009 Macmillan Publishers, Ltd.
The data suggest that CoCr nanoparticle-induced mitochondrial toxicity in BeWo cells leads to a pannexindependent activation of P2 receptors. Subsequent ATPand Ca2+-dependent signaling is responsible for the DNA damage in the underlying fibroblasts. Thus, nanoparticles can induce toxicity across an epithelial barrier, and in fact, Published online 02/15/2010 • DOI: 10.1021/tx900455s $ © 2010 American Chemical Society
Spotlight the cells of the barrier may play an active role in the toxic mechanism. These findings suggest that future investigations of nanoparticle toxicity must take into account how complex tissue and organ structures influence the effects of these materials. • Carol A. Rouzer
Pore-Forming Neurotoxins Deposition of protein aggregates is a well-recognized finding in neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Recent work has suggested, however, that neurotoxicity is not caused by the insoluble aggregates but rather by soluble protein oligomers formed during the aggregation process. Study of these oligomers is difficult because of the fact that they are short-lived intermediates that cannot be isolated in sufficient concentration for characterization. Now, Kim et al. [(2009) J. Am. Chem. Soc. 131, 17482] have solved this problem in the case of R-synuclein (RS), a key component of protein aggregates found in Parkinson’s disease.
Adapted from Kim et al. [(2009) J. Am. Chem. Soc. 131, 17482]. Copyright 2009 American Chemical Society.
Kim et al. subjected preparations of RS amyloid fibrils to supercooling at -13 °C for 48 h, which led to cold-induced dissociation of the fibrils. NMR and fluorescence analysis indicated that the dissociated samples contained approximately 15% monomers, 20-30% fibrils, and 55-65% spherical soluble oligomers. Centrifugation yielded concentrated preparations of pure oligomers. The oligomers did not stain with the amyloid-specific dye ThT, but they were recognized by the A11 antibody directed against a structural feature of neurotoxic proteins. These observations, combined with the ability of the oligomers to facilitate monomer aggregation into fibrils, suggested that these soluble species are a neurotoxic aggregation intermediate of RS. The concentrated preparations of RS oligomers allowed Kim et al. to demonstrate that these species form pores in a variety of artificial membranes. The well-defined conductance states of the pores were similar to those observed with other pore-forming peptides. Structural elucidation by NMR indicated that the β structure of the oligomers was not fibrillar and was distinct from that of either the monomers or the amyloid fibrils. The data support the value of cold-induced dissociation to generate concentrated samples of RS oligomers for further study and suggest that pore formation and a
Published online 02/15/2010 • DOI: 10.1021/tx900455s © 2010 American Chemical Society
$
nonfibrillar structure may be important features related to the neurotoxicity of protein aggregates. • Carol A. Rouzer
Antioxidant tRNA Misacylation Methionine residues are known to protect proteins against oxidative damage, so the findings by Netzer et al. [(2009) Nature 462, 522] that oxidative stress increases misacylation of Met into noncognate tRNAs is of great interest. Netzer et al. devised a microarray-based method to measure tRNA misacylation, using an array that detects 274 cytosolic human tRNAs as 42 families and all 22 human mitochondrial tRNAs. Pulse-labeling HeLa cells (or hepatocytes in a living mouse) with [35S]-Met revealed the incorporation of approximately 1.5% of radioactivity into 8 families of noncognate cytosolic tRNAs. Control experiments ruled out incorporation by peptidylation rather than acylation, incorporation of impurities from the [35S]-Met, and incorporation of metabolites of [35S]-Met. No other amino acids exhibited misacylation, and no mitochondrial tRNAs were involved. Infection of HeLa cells with influenza A virus increased Met misacylation to 13%, with 18 new families affected. Similar results were observed with vaccinia virus and adeno virus or by exposing cells to poly-IC or bacterial lipopolysaccharide, which activate toll-like receptors 3 and 4, respectively. Similarly, exposure of HeLa cells to inducers of the unfolded protein response (tunicamycin and MG132) and growing cells past confluence resulted in increased Met misacylation, although heat shock-induced stress had the opposite effect. The purified Met-tRNA synthetase complex, consisting of Met-tRNA synthetases and 8 other synthetases, misacylated tRNAs in vitro (including Lys tRNA), with greater rates of misacylation occurring for the polysome-associated enzyme; however, Lys-tRNA synthetase did not misincorporate Met into Lys tRNA. The observation that the rates of disappearance of misacylated tRNAs and Met-tRNAs were similar during a chase with cold Met and that the rates were similarly affected by cycloheximide suggested that the misacylated tRNAs were used for protein synthesis. Aberrant incorporation of Met into a ubiquitin reporter protein in virus-infected HeLa cells further supported this conclusion. Viral and inflammatory stimuli often increase oxidant stress in cells. Netzer et al. showed that the oxidants arsenite, telluride, and H2O2 are strong inducers of Met misincorporation and that this effect is blocked by inhibitors of NADPH oxidase. Together, the data support the hypothesis that oxidative stress leads to an increased misincorporation of Met into noncognate tRNAs with a consequent increase in Met incorporation into proteins, a response which may protect cells against oxidative protein damage. • Carol A. Rouzer TX900455S
Vol. 23,
No. 2, •
CHEMICAL RESEARCH IN TOXICOLOGY 297
