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BENZO[A]PYRENE-MEDIATED ADRENERGIC SIGNALING Polycyclic aromatic hydrocarbons (PAHs) are common toxicants resulting from incomplete combustion of organic materials. Their toxicity primarily results from their ability to activate the aryl hydrocarbon receptor (AhR), which induces expression of genes bearing xenobiotic response elements, among them metabolic enzymes such as cytochromes P450 1A1 and 1B1. PAHs also induce a transient increase in intracellular Ca2+, which is independent of, but required for, AhR activation. The exact mechanism of this Ca2+ signal is not known, but evidence that PAHs interact with the β2-adrenergic receptor (β2ADR) led Mayati et al. [(2011) J. Biol. Chem., 287, 4041] to investigate its role in PAH-dependent AHR activation. The prototypical PAH benzo[a]pyrene (B[a]P) elicited an increase in [Ca2+]i in human microvascular endothelial cells (HMEC-1) with a time course similar to that of the β2ADR agonist salbutamol. The response to B[a]P was blocked by the β2ADR antagonist ICI-118,551, the nonspecific βADR blockers propranolol and carazolol, an antibody against the β2ADR, and siRNA-mediated suppression of β2ADR protein expression. β2ADR activation leads to G protein-dependent stimulation of adenylate cyclase (AC), resulting in increased intracellular cAMP. Consistently, B[a]P exposure led to an elevation of cAMP in HMEC-1 cells, which was blocked by ICI-118,551 and the AC inhibitor dd-Ado. Inhibition of AC by dd-Ado or the G protein blocker suramin suppressed the [Ca2+]i increase in response to B[a]P. cAMP activates the guanine nucleotide exchange factors Epac-1 and Epac-2. Both the Epac inhibitor BFA and the siRNAmediated suppression of Epac-1 expression led to a reduced [Ca2+]i response to B[a]P in HMEC-1 cells. Epac-1, in turn, activates phospholipase C, leading to increases in intracellular inositol tris-phosphate (IP3), a mobilizer of [Ca2+]i. Mayati et al. showed that L-690.333, LiCl, 2-ABP, and TMB-8, all inhibitors of IP3-generation and/or IP3-mediated [Ca2+]i mobilization, blocked the response to B[a]P. Inhibitors of β2ADR-dependent signaling, including ICI-118,551, propranolol, suramin, dd-Ado, and BFA, all suppressed the B[a]P-mediated increase in P450 1B1 expression in HMEC-1 cells. Together, the results suggest that B[a]P activation of the AhR requires a [Ca2+]i signal that is propagated by the β2ADR. The finding that B[a]P specifically binds to the β2ADR with a Kd of 10 nM further supports this conclusion. Carol A. Rouzer
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NANOTOXICITY: DIVIDED WE STAND
concentrations led to increased particle uptake, the apparent saturation was not due to the inability of the cells to take up more particles. Kim et al. proposed that the saturation of nanoparticle uptake was due to a reduction in the number of particles per cell due to cell division. A computational simulation supported this hypothesis. The combined use of 7-aminoactinomycin D to quantify cellular DNA and ethynyl deoxyuridine to label cells undergoing DNA synthesis allowed Kim et al. to sort cells according to their phase of the cell cycle. They found that nanoparticle content decreased with cell cycle phase in the order of G2/M > S > G0/G1, although the rate of nanoparticle uptake was similar regardless of cell cycle phase. Cells preloaded with nanoparticles and then placed in particle-free medium showed a gradual decrease in nanoparticle content for the population as a whole. However, when separated into populations of cells that just completed division versus all other cells, the particle content remained stable for both populations, with the quantity of particles in the recently divided cells about half of that in the remaining population. Kim et al. conclude that cells take up nanoparticles as they progress through G0/G1 to S and then G2/M. However, during mitosis, the particles are divided between the two daughter cells, halving the content per cell on average. The results imply that nonlinear uptake should not be mistaken for particle export and that nanoparticle burden will be lower in cells that are rapidly dividing. It also suggests that cells in different phases of the cell cycle could have different responses
Reprinted with permission from Macmillan Publishers Ltd. from Kim et al. (2011) Nat. Nanotechnol. 7, 62. Copyright 2011. The complexity of the interactions of nanoparticles with cells complicates the elucidation of nanoparticle toxicity. Nanoparticle properties such as size, aggregation state, and surface functionalization all influence the cellular response. Now, Kim et al. [(2011) Nat. Nanotechnol. 7, 62] show that the cell cycle also plays a role in nanoparticle uptake and potentially toxicity. Kim et al. used carboxylated polystyrene nanoparticles bearing a fluorescent dye, which allowed the quantification of intracellular particle load. Exposure of A549 human lung carcinoma cells to these particles for up to 72 h induced no acute toxicity and had no effect on the cell cycle. Upon continuous exposure, cells took up the nanoparticles into lysosomes as indicated by colocalization with lysosomalassociated membrane protein 1 by immunofluorescence. The uptake of the nanoparticles was linear initially but then leveled off after about 1 day, despite the ongoing presence of particles in the culture medium. Because higher nanoparticle © 2012 American Chemical Society
Published: March 19, 2012 586
dx.doi.org/10.1021/tx3000413 | Chem. Res. Toxicol. 2012, 25, 586−587
Chemical Research in Toxicology
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reduced phosphatase activity allows increased phosphorylation and activation of PERK, promoting the ER stress response. Carol A. Rouzer
to nanoparticles. These are important considerations for nanotoxicologists. Carol A. Rouzer
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H2S ACTIVATES THE ER STRESS RESPONSE
DNA REPAIR CENTERS AND CANCER RISK LINEARITY Within minutes of ionizing radiation (IR) exposure, DNA double strand breaks (DSBs) become the site of assembly of DNA damage recognition and repair proteins. These sites, known as radiation-induced foci (RIF) can be detected by immunofluorescence staining. While the number of DSBs increases linearly with IR dose, attempts to demonstrate a similar linearity between dose and RIF have led to inconclusive results. Realizing that measurement of RIF at any single time point fails to account for the dynamic formation and resolution of the sites, Neumaier et al. [(2011) Proc. Natl. Acad. Sci. U.S.A., published Dec. 19, DOI: 10.1023/pnas.1117849108] developed a mathematical model to extract the absolute number of RIFs from kinetic data acquired at multiple time points following IR exposure. Neumaier et al. used MCF10A cells transiently transfected with 53BP1-GFP, a fluorescently labeled protein that accumulates in RIF. Live cell imaging of these cells following varying doses of IR exposure allowed the investigators to monitor RIF formation and resolution in real time. Application of the mathematical model to these data revealed that that the total number of RIF/dose decreased with increasing IR exposure. At higher doses, RIF appeared more quickly and resolved more slowly. Also, while the size did not vary, the intensity of RIF was greater at higher doses. Similar results emerged from experiments using fixed MCF10A cells immunostained for 53BP1. The number of RIF/Gy was 4-fold greater in cells receiving 0.1 Gy of IR than those receiving a 2 Gy dose. The rate of RIF formation at the higher dose was twice as fast as at the lower dose, but the resolution rate was 5-fold higher at the lower dose. Treatment of the cells with an inhibitor of the DNA damage response kinase ATM, prior to IR treatment resulted in a lower number of RIF at all doses, but the pattern of differences between low and high doses of IR remained unchanged. Treatment of cells with high-energy iron ions resulted in a defined linear track of high-energy DNA damage surrounded by a broader region of lower energy damage, providing a direct comparison of the effects of high- versus low-dose damage in a single nucleus. The results were consistent with those obtained with IR treatment. RIF appeared more quickly, resolved more slowly, and were more intense in regions of high-energy as compared to low energy damage. Neumaier et al. propose that the lack of linearity of RIF with dose could be due to the existence of DNA repair centers with a minimum interdistance of 1 μm. As the IR dose increases, the likelihood that more than one DSB will be present in a single center increases. Rapid DSB clustering would explain the increased rate of RIF formation and the higher RIF intensity at high IR dose. Also, the presence of multiple DSBs in one site explains the slower RIF resolution under these conditions. The presence of multiple DSBs in one site increases the chance of toxic chromasomal rearrangements. If true, neglecting the impact of the microenvironment and immune response on damaged cells in a full organism, extrapolation of cancer risk linearly from high doses of IR as is currently done, could then overestimate the risk at lower doses. Carol A. Rouzer
Reprinted with permission from Krishnan et al. (2012) Sci. Signal. 4, ra86. Copyright 2011 AAAS. Cells use the endoplasmic reticulum (ER) stress response to combat the deleterious effects of abnormally folded proteins that may form upon toxicant exposure. Thus, the report from Krishnan et al. [(2011) Sci. Signal. 4, ra86] that the ER stress response is regulated by H2S is of particular interest. H2S, a recently recognized gaseous signaling molecule, is formed by the action of cystathionine β-synthase and cystathionine γ-lyase (CSE) on cysteine. H2S signaling is mediated by sulfhydration of specific cysteine residues in target proteins, leading Krishnan et al. to explore its effects on protein tyrosine phosphatase (PTP)1B. Like all PTPs, PTP1B contains a critical active site cysteine (Cys-215), and the enzyme is inactivated by oxidation or nitrosylation of this residue. Incubation of PTP1B with H2S caused a concentration- and time-dependent inactivation with a second-order rate constant 10fold higher than those for inactivation by H2O2 or NO. MS analysis confirmed that H2S exposure resulted in sulfhydration of Cys-215. Incubation of sulfhydrated PTP1B with DTT, GSH, or thioredoxin plus thioredoxin reductase (TR/TRR) restored enzyme activity. A biotinylated iodoacetic acid probe and MS analysis revealed that incubation of HEK293 cells with H2S resulted in sulfhydration of intracellular PTP1B at Cys-215. Treatment of HeLa cells with tunicamycin to induce ER stress led to increases in endogenous H2S levels, the sulfhydration of PTP1B, and a reduction of PTP1B activity in the immunopreciptiated enzyme. Stable expression of an shRNA directed against CSE resulted in decreased CSE expression, reduced H2S formation in response to tunicamycin, and a reduction in PTP1B sulfhydration with preservation of enzymatic activity. Treatment of HeLa cells with tunicamycin led to increased phosphorylation of a 150 kD protein that was identified as the ER stress response protein PERK. This was not observed in cells with reduced CSE expression. PERK activation requires autophosphorylation of Tyr-619, which then promotes phosphorylation of Thr-980. Krishnan et al. showed that an inactive mutant PTP1B protein immunoprecipitates PERK from tunicamycin-treated cells. This was not true of a Y619F mutant PERK. These results confirm that tunicamycin treatment leads to phosphorylation of Tyr-619 of PERK and that the phosphorylated protein is a substrate of PTP1B. The results suggest that ER stress leads to CSE-dependent production of H2S, which inactivates PTP1B. The resulting 587
dx.doi.org/10.1021/tx3000413 | Chem. Res. Toxicol. 2012, 25, 586−587
