In This Issue pubs.acs.org/crt
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SPECIAL FEATURES The year 2013 ushers in a new era for CRT as it welcomes its new Editor-in-Chief, Steve Hecht. His inaugural editorial (DOI: 10.1021/tx300497e) provides a vision for the future of the journal and the broader field of chemical toxicology. We also welcome Lisa Peterson as CRT’s new Associate Editor and draw your attention to her comprehensive review of the mechanisms of toxicity of furan-containing compounds, which appears in this issue (DOI: 10.1021/tx3003824).
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DETOXICATING QUARTZ WITH CARBON
Inhibition of nitric oxide and tumor necrosis factor-α release by MH-S cells in response to carbon-coated as compared to uncoated quartz indicated that the coated particles did not induce the inflammatory response typically observed in quartztreated cells. In all of these assays, 1% and 10% coated particles invoked similar cellular responses. Mixtures of quartz and carbon that had not been milled elicited responses similar to those of uncoated quartz. Together, the results suggest that carbon in intimate association with quartz markedly reduces quartz-mediated toxicity, likely by suppressing the surface reactivity of the quartz particles. These interesting findings may help to explain why different forms of quartz vary substantially in their toxicity.
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Although the International Association for Research on Cancer (IARC) classified quartz dust as a Group 1 human carcinogen, it acknowledged that the carcinogenicity of quartz depends on intrinsic characteristics of the material and/or external factors that are not well understood. Despite containing substantial amounts of quartz, coal dust is not a known carcinogen. These observations led Ghiazza et al. (DOI: 10.1021/tx300299v) to investigate the effects of intimately associated carbon on quartz toxicity. Ghiazza et al. combined commercial quartz dust with 1% or 10% carbon soot and then milled the mixtures to form quartz particles with a tightly adherent coating of carbon. The carbon coating had no effect on quartz particle size or shape, though the 10% coating caused a significant increase in surface area, consistent with the larger surface area of the constituent carbon particles. The 10% carbon coating also decreased the negative surface charge of the quartz particles. Uncoated quartz particles generated hydroxyl radicals and carbon-centered radicals when incubated with H2O2 or formate ions, respectively. Coating with either 1% or 10% carbon completely suppressed radical formation under these conditions. EPR spectroscopy indicated the presence of siliconand oxygen-centered radicals in the quartz particles, while only carbon-centered radicals were detected in the carbon-coated particles. Carbon coating significantly reduced the cytotoxicity of the quartz particles to MH-S murine lung macrophages, as indicated by lactate dehydrogenase release. The carbon coating also suppressed quartz-mediated generation of thiobarbituric acid-reactive substances, a measure of oxidative stress. © 2013 American Chemical Society
BIOACTIVATION OF CAPSAICIN
Capsaicin and related compounds provide the pungency to hot peppers and are used in a number of consumer products and medications. Topical use and ingestion of capsaicin (0.5 to 4 mg/kg/day) produces high local and moderate circulating concentrations. Extensive P450-mediated metabolism of capsaicin results in a relatively short half-life, and analysis of the metabolites indicates that the alkyl side chain, aromatic ring, and its substituents are all targets. Prior studies have shown that P450-dependent metabolism of capsaicin is associated with genotoxicity, and the finding of some GSH conjugates in metabolic studies suggests the formation of reactive species. This led Reilly et al. (DOI: 10.1021/tx300366k) to embark on a full characterization of potentially toxic capsaicin metabolites. Reilly et al. incubated capsaicin and structural analogues with human liver microsomes or individual P450 enzymes in the presence of GSH or β-mercaptoethanol (BME) as electrophile trapping agents. A combination of LC-MSn, NMR, 18O incorporation from H218O, and chemical synthesis resulted in the characterization of nine GHS conjugates, with structural Published: January 18, 2013 2
dx.doi.org/10.1021/tx300498x | Chem. Res. Toxicol. 2013, 26, 2−3
Chemical Research in Toxicology
In This Issue
confirmation derived from MSn and NMR analysis of the GHS and corresponding BME conjugates (see the Figure). The most abundant GSH conjugate was derived from a quinone methide or o-quinone generated by O-demethylation of the methoxy group at C3 of the aromatic ring (see the Figure). GSH addition to the aromatic ring of this electrophile produced metabolite G2, which comprised 71% of the total conjugates observed in vitro using human liver microsomes. Attack of GSH at the benzylic carbon of the same intermediate yielded the minor conjugate G1. Oxidation of the aromatic ring at C6 followed by GSH adduction at another aromatic ring position yielded the second most prevalent GSH conjugate, G9, at 17% of the total. Similar aromatic ring adduction following hydroxylation at C5 produced metabolite G8 (3.4% of the total), while GSH addition to the benzylic carbon of the C5hydroxylated capsaicin formed a minor metabolite (G7). The third most prevalent metabolite (G3 at 5% of the total) resulted from GSH addition to the benzylic carbon following oxidation of capsaicin to a quinone methide. Minor metabolites (G4, G5, and G6) resulted from GSH addition to the aromatic ring of this same quinone methide intermediate. Finally, identification of a capsaicin dimer suggested reaction between two capsaicin radical metabolites. The results confirmed the formation of multiple reactive electrophiles generated through P450-mediated metabolism of capsaicin, alternative capsaicinoid analogues, and structurally related vanilloid ring-containing compounds. Most of the conjugates were produced by P450 1A2, 2C19, 2D6, and 3A4. Administration of capsaicin to mice confirmed the formation of metabolites G2, G3, G6, and G8 in vivo. Previous reports suggested that capsaicin can act as a cancer chemopreventive agent or as a cocarcinogen within the same model system. Reilly et al. conclude that these seemingly contradictory effects are likely the result of the combination of relative degrees of metabolic destruction, bioactivation, and TRPV1 receptor activation in each individual target tissue.
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Initial studies indicated that the LC50 (concentration causing death of 50% of cells) of arsenite in RIMM-18 cells was 13.6 μM. All subsequent studies used arsenite at 500 nM to avoid overt cytotoxicity. Tokar et al. discovered that long-term arsenite exposure (28 weeks) induced a 2- to 4-fold increase in the secretion of matrix metalloproteinases-9 and -2, a 12-fold increase in the cells’ ability to form colonies in soft agar, a 2fold increase in invasiveness, and a marked increase in the ability to form nonadherent spheres with large, branching, ductlike structures in Matrigel (see the Figure). All of these changes are consistent with malignant transformation and the development of an aggressive phenotype. The arsenite-mediated changes in morphology and growth pattern were accompanied by alterations in gene expression, including a 7-fold increase in the expression of cyclooxygenase2 (Pghs-2), an enzyme strongly associated with the malignant phenotype. Expression of Wnt4 and Wilm’s tumor protein-1 (Wt-1) first decreased and then increased, while that of bone morphogenic protein-7 (Bmp-7) and β-Catenin decreased and remained low. All of these proteins are important for normal kidney development. Wt-1, which acts as both a tumor suppressor and an oncogene, plays a role in the development of Wilm’s tumor, a childhood cancer of the kidneys. Increased expression of kidney SC/PC markers, Cd24, Osr1, Cd133, and Ncam, in arsenite-treated cells provided further confirmation of the importance of this cell population in the response to the metalloid. The cells adapted to the presence of arsenite by increased expression of metallothionein-1 and -2 (Mt-1 and Mt-2), superoxide dismutase (Sod-1), and the ATP binding cassette transporter C2 (Abcc2). The metallothioneins bind arsenic, while the latter two proteins are involved in the cellular stress response. Together, the data support the hypothesis that long-term exposure of renal SC/PC cells to arsenite results in physiological changes consistent with malignant transformation. These observations suggest that the carcinogenicity of arsenite may be primarily targeted to renal SC/PCs. The results also reveal the value of RIMM-18 cells as a useful model system to more extensively explore the chemical mechanisms underlying arsenite’s effects on the kidney.
STEM CELL-TARGETED CARCINOGENICITY
The presence of arsenic in drinking water presents a worldwide public health problem. Classified by the International Association for Research on Cancer (IARC) as a known human carcinogen, arsenic targets multiple sites, including skin, bladder, liver, and lung. People exposed to arsenic early in life exhibit increased risk of mortality from cancer of the kidney, and animal models indicate that exposure to arsenic in utero predisposes one to renal cancer in adulthood. These findings suggest that arsenic may act by altering a long-lived stem or progenitor cell (SC/PC) population during early life exposure and that these abnormal SC/PCs play a role in tumorigenesis later in life. To test this hypothesis, Tokar et al. (DOI: 10.1021/tx3004054) investigated the effects of sodium arsenite exposure on the RIMM-18 kidney SC/PC cell line, which was derived from rat metanephric mesenchyme. 3
dx.doi.org/10.1021/tx300498x | Chem. Res. Toxicol. 2013, 26, 2−3
