In this issue Butadiene Mutagenicity 1,2,3,4-Diepoxybutane (DEB) is the primary carcinogenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant that is derived from automobile emissions and cigarette smoke. As a bis-electrophile, DEB can react with multiple sites in DNA, and its ability to generate DNA-DNA interstrand cross-links is well established. However, DEB’s potential to form exocyclic adducts has not yet been fully explored. Since these bulky lesions often lead to mispairing during DNA synthesis, Seneviratne et al. (p 118) hypothesized that an exocyclic adduct with deoxyadenosine might explain the frequent A to T transversion mutations observed with DEB exposure. They now present data to support this hypothesis.
Reaction of DEB with nucleotides produces 2-hydroxy-3,4-epoxybut-1-yl lesions. Seneviratne et al. generated this lesion (compound 1) at the N6 position of dA through the reaction of 6-chloropurine deoxyriboside with 1-amino-2-hydroxy-3,4-epoxybutane. This reaction also generated an exocyclic adduct (compound 2). Compound 1 decomposed in aqueous solution to yield two additional exocyclic adducts (3 and 4). Mass spectral analysis indicated that the molecular 2
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mass of all three exocyclic adducts corresponded to the addition of DEB to dA; however, the fragmentation patterns implied unique structures for each. UV spectroscopy suggested that 2 was an N6,N6-adduct of dA, while 3 and 4 were both 1,N6-adducts. Periodate oxidation revealed that 2, but not 3 or 4, contained a vicinal diol. The exact structures of multiple stereoisomers of each compound were elucidated through 1- and 2-dimensional 1H- and 13C NMR and total stereospecific synthesis. DEB occurs as three stereoisomers, R,R, S,S, and meso. All three are produced by metabolism of 1,3-butadiene in vivo. Incubation of calf thymus DNA with each DEB isomer individually, followed by hydrolysis, led to the isolation of distinct stereoisomers of compounds 3 and 4, with 4 predominating. The three DEB stereoisomers were equal in their ability to generate the exocyclic adducts. Compounds 3 and 4 were also identified in hydrolysates of DNA obtained from the livers of mice treated with 1,3-butadiene by inhalation. The adduct stereoisomers present in these samples suggested that the source was primarily R,Rand/or S,S-DEB rather than the meso compound, likely due to different rates of formation or degradation of each DEB isomer in vivo. The formation of compound 3 from the decomposition of 1 in vitro was explained by an intramolecular nucleophilic substitution, but this was not the case for compound 4. Indeed, kinetic analyses suggested that compound 4
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was formed from 3. Further studies showed that this conversion could occur slowly under physiological conditions via a Dimroth rearrangement, with the equilibrium favoring the more stable 4. However, Seneviratne et al. propose that, in vivo, compound 4 is derived from the nucleophilic attack of DEB at the N-1 position of dA followed by cyclization, while compound 3 results from initial attack at the N6 position, or from rearrangement of compound 4. Notably, compound 2 appears to only occur in substantial quantities in nonaqueous environments. These data clearly indicate the potential of DEB to form exocyclic adducts with dA. The lesions were not repaired by base excision or nucleotide excision repair; therefore, they may remain in the genome for sufficiently long periods of time to be a cause for the A to T transversions associated with 1,3-butadiene exposure. Proteomics of Sulfur Mustard Sulfur mustard (SM) has been used as a chemical
warfare agent since the First World War. Although much is known about changes in individual metabolic and signaling pathways in cells exposed to SM, we lack a clear understanding of how these changes integrate to generate the full toxic response. Such knowledge would be useful to design therapies for SM poisoning. Now Everley and Dillman (p 20) apply a proteomics approach based on stable isotope labeling with amino acids in cell culture (SILAC) to identify large-scale changes in protein phosphorylation in SM-exposed HaCaT cells (a human keratinocyte model).
Everley and Dillman first incubated HaCaT cells with 12 C614N4-Arg and 12C614N2Lys (control cells) or 13C615N4-
Special Features Cardiotoxicity limits the clinical use of the quinone-hydroquinone anthracycline antitumor agents, such as doxorubicin. Reduction of the quinone followed by redox cycling is widely believed to cause toxic damage to cardiomyocytes. Now Editorial Advisory Board member Giorgio Minotti (Menna et al., p 6) describes a mechanism by which H2O2 generated during redox cycling, combined with oxyferrous myoglobin, may lead to oxidation of the anthracycline and its ultimate degradation. Do not miss this opportunity to get a new perspective on the mechanism of anthracycline cardiotoxicity. Published online 01/18/2010 •
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In this issue Arg and 13C615N2-Lys (experimental cells) for five passages to completely label cellular proteins with the respective amino acids. The experimental cells were then exposed to 200 µM SM for 20 min, a treatment known to induce inflammatory signaling. Lysates from control and experimental cells were combined, and proteins were fractionated by gel electrophoresis. Following in-gel digestion, phosphopeptides were isolated by immobilized metal ion affinity chromatography (IMAC). Mass spectrometric analysis of the phosphopeptides revealed SMdependent changes in phosphorylation by differences in the signal intensity for heavy versus light peptides derived from experimental versus control cells, respectively. This approach identified 2315 unique phosphorylation sites on 790 proteins, implicating the DNA damage, apoptotic, NF-κB, and RhoGTPase signaling pathways. Of the 790 proteins, 86 exhibited a 2-fold or greater change in phosphorylation levels in response to SM. De novo network mapping using Ingenuity Pathway Analysis revealed associations between 27 of the 86 proteins. Although some of these SM-induced changes have been previously identified, a large number of new interactions were defined in this report. Thus, the SILAC/IMAC approach promises to aid in the discovery of new targets for SM therapy. Carcinogenicity of Snuff Smokeless tobacco is considered by some to be a
safe alternative to smoking. However, the use of smokeless tobacco is associated with oral, pancreatic, and esophageal cancers, and the International Agency for Research on Cancer lists 28 carcinogens present in these products. Tobaccospecific nitrosamines are considered the most important of these carcinogens. Since smokeless tobacco is not burned during consumption, there is a widespread assumption that its use does not expose the consumer to significant levels of polycyclic aromatic hydrocarbons (PAHs). The error in this assumption has now been revealed by Stepanov et al. (p 66) who have developed a single gas chromatography-mass spectrometry assay for 23 PAHs and used that assay to determine levels of these compounds in 23 brands of moist snuff and 17 brands of spitless tobacco.
Moist snuff comprises >80% of smokeless tobacco sold in the U.S. All but one of the varieties tested contained relatively high levels of total PAHs (11.6 ( 3.7 µg/ g) with naphthalene, a class 2B carcinogen, being the predominant component (15% of the total). In contrast, spitless tobacco products contained much lower PAH levels (1.28 ( 0.28 µg/ g), although naphthalene predominated in these samples as well at 85% of the total. Stepanov et al. point out that tobacco for moist snuff
Published online 01/18/2010 • DOI: 10.1021/tx900416t © 2010 American Chemical Society
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is fire cured, whereas that for spitless tobacco is pasteurized. Exposure to wood smoke during curing is the likely source of PAHs in moist snuff, and the fact that one brand contained much lower quantities of PAHs indicates that changes in manufacturing processes could drastically reduce levels in other brands as well. However, moist snuff and spitless tobacco both contain similar amounts of naphthalene, suggesting that this PAH can not readily be eliminated by changes in curing method alone. Thus, PAHs are a significant class of carcinogens in smokeless tobacco.
Smith et al. loaded the silicone with polycyclic aromatic hydrocarbons (PAHs) as model hydrophobic compounds by partitioning from a methanol solution followed by the removal of the methanol with water. This procedure gave reproducible and stable silicone concentrations. PAH release from the loaded silicone into aqueous solutions was rapid and reproducible, with equilibrium partitioning being reached within hours. The buffering capacity of the O-rings was sufficient to maintain stable concentrations over more than 72 h for most compounds.
Better Toxicity Testing The costs and ethical controversies surrounding animal experimentation have led to an increased focus on in vitro toxicity testing. However, a technical challenge arises when testing hydrophobic compounds on cells maintained in tissue culture well plates. For these compounds, a high susceptibility to losses via volatilization or sorption to plastic and medium components make maintaining defined and constant cell exposure concentrationsdifficult.Now Smith et al. (p 55) address this problem using a silicone O-ring passive dosing format aimed at the routine in vitro testing of hydrophobic chemicals. Partitioning of preloaded test compounds from biologically inert silicone into the culture medium compensates for any losses and gives well-defined and constant freely dissolved exposure concentrations.
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The O-ring passive dosing format was applied to test a range of PAHs at their aqueous solubility in an array of in vitro cell culture assays. Only those PAHs with highest chemical activities stimulated the formation of reactive oxygen species and MCP-1 secretion, while inhibiting the induction of the IL-8 cytokine promoter. Smith et al. conclude that passive dosing using silicone O-rings is a simple, practical, and effective technique for achieving defined and constant exposure of hydrophobic compounds in cellular assays, and could play an important role in the testing of mixture toxicity. TX900416T
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