In This Issue pubs.acs.org/crt
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SPECIAL FEATURES While the structural and catalytic roles of protein thiols have been appreciated for many years, the importance of these functionalities in redox signaling is just now being realized. Jacob et al. (DOI 10.1021/tx200342b) provide a comprehensive look at the complex chemistry of oxidative cysteine postranslational modifications and share their perspective on how these reactions have become a focal point for therapeutic and toxicologic research. As the industrial applications for nanoparticles continue to grow, concern about the toxicity of these new materials increases. The study of nanotoxicology offers unique challenges, particularly with regard to particle characterization, aggregation, and solubility. Now, Horie et al. (DOI 10.1021/tx200470e) offer a thorough review of primary mechanisms of nanoparticle toxicity and important considerations for the design of nanotoxicologic evaluations.
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RADIOACTIVITY IN DRUG SAFETY ASSESSMENT
Although the use of radiolabeled compound combined with LC or LC/MS to identify drug metabolites is familiar to many, the techniques of whole body autoradiography and quantitative whole body autoradiography are less well-known. Solon provides an excellent review (DOI 10.1021/tx200509f) of these approaches, including their role in drug discovery and compound safety assessment. For an excellent example of how a radiolabel can be used to dissect the mechanism of compound toxicity, see the article by Zhang et al. (DOI 10.1021/tx200524d). Their application of radiolabeled compound in an elegant series of experiments led to an understanding of the compound’s adrenotoxicity and the means to eliminate the toxicity as drug development progressed. Of course, a special issue on the use of radiolabel would not be complete without consideration of alternatives. To this end, Mutlib et al. (DOI 10.1021/tx2005629) provide convincing data suggesting that, for fluorinated compounds, 19F-NMR may replace the use of radiolabel, at least for some ADME studies. Together, these articles provide a great introduction to the use of radioactivity in drug discovery. Watch for additional papers on the subject in future issues of CRT!
The use of radiolabeled compound is an intrinsic and currently irreplaceable part of the drug discovery and development process. In most cases, it remains the best way to completely assess the absorption, distribution, metabolism, and excretion (ADME) properties of new molecular entities. Yet, the expense of synthesizing radiolabeled compounds, the drive to reduce the production of radioactive waste, and the effort to decrease the use of laboratory animals combine to challenge the pharmaceutical industry to revise or replace the use of radioactivity. In this climate, Editorial Advisory Board member Abdul Mutlib suggested that a special issue on the use of radioactivity in drug safety assessment would be timely. The result is this collection of articles edited by Associate Editor Fred Guengerich who also provides an introduction (DOI 10.1021/tx3000522) to the subject. For a broad and comprehensive overview of the use of radiolabel in the drug development process, the best place to start is with the review by Penner et al. (DOI 10.1021/tx300050f). They describe the different types of questions that are addressed by radiolabel studies, the advantages and pitfalls of the approach, and issues surrounding experimental design and execution. Because of the importance of the radiolabeled compound at multiple stages of drug discovery, many pharmaceutical companies have dedicated isotope chemistry laboratories. Isin et al. (DOI 10.1021/tx2005212) provide the perspective of the isotope laboratory at AstraZeneca with regard to the application of radiolabeled compound in the drug development process. They include illustrative examples of how the approach has yielded important insight leading to the decision to advance or abandon compounds at key points in development. © 2012 American Chemical Society
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GSH CONJUGATION = BUTADIENE ACTIVATION
Butadiene is an industrial chemical widely used in the manufacture of plastics. It is also an environmental toxicant present in cigarette smoke and automobile exhaust. A mutagen, and likely human carcinogen, butadiene reacts with DNA following epoxidation by cytochrome P450 enzymes. The most mutagenic metabolite is 1,2,3,4-diepoxybutane (DEB), due to its high electrophilicity and bifunctionality. DEB occurs in three stereoisomeric forms, S,S-, R,R-, and meso-, in order of decreasing mutagenicity, although the potency differences are not large. Although conjugation with GSH is generally regarded as a detoxication mechanism, conjugates of DEB with GSH Published: March 19, 2012 584
dx.doi.org/10.1021/tx3000544 | Chem. Res. Toxicol. 2012, 25, 584−585
Chemical Research in Toxicology
In This Issue
and must be reduced to the corresponding methyl adducts prior to quantification. This makes it difficult to distinguish formaldehyde-generated adducts from those resulting from direct methylation. Now, Lu et al. (DOI 10.1021/tx200426b) use isotopically labeled formaldehyde and alkylating agents to distinguish unequivocally the adducts resulting from exogenous versus endogenous sources of formaldehyde (see figure). MS analysis using selected reaction monitoring of reduced DNA isolated from control cells in culture revealed transitions at m/z 282.2→m/z 166.1 and m/z 266.2→m/z 150.1. These transitions corresponded to N2-methyl-dG and N6-methyl-dA, respectively. The fact that the sizes of the peaks were markedly increased by sample reduction indicated that they originated from the corresponding hydroxymethyl adducts formed from the attack of endogenous formaldehyde on DNA. Analysis of reduced DNA following exposure of cells to [13CD2]-formaldehyde resulted in a new peak at m/z 285.2→m/z 169.1, indicating the formation of [13CD2]-N2-hydroxymethyl-dG. However, no corresponding peak for [13CD2]-N6-hydroxymethyl-dA was detected. Careful control studies indicated that deuterium exchange of the isotopically labeled adducts occurs during prolonged cell culture, but even when this possibility was taken into account, no dA adduct formation from the exogenous formaldehyde could be demonstrated. Cytochrome P450 2E1-mediated metabolism of N-nitrosodimethyl-D6-amine (D6-NMDA) yields D2-formaldehyde and a diazonium ion which then forms the carbonium ion CD3+ (see figure). Incubation of cells with D6-NMDA followed by reduction, DNA isolation, and MS analysis revealed a peak at m/z 284.2→m/z 168.1 consistent with the formation of the D2-N2-hydroxymethyl-dG adduct from D2-formaldehyde, but no D2-N6-hydroxymethyl-dA formation was observed. Peaks at m/z 285.2→m/z 169.1 and m/z 269.2→m/z 153.1 indicated the formation of D3-N2-methyl-dG and D3-N6-methyl-dA from direct methylation of each base. These latter peaks were also detected following incubation of cells with D3-methylmethanesulfonate, which is a methylating agent only. Incubation of cells with methylnitrosourea also led to the formation of N2-methyl-dG and N6-methyl-dA, but these adducts were formed in much lower quantities than N7-methyl-dG and O6-methyl-dG. The results indicate that, while endogenous formaldehyde reacts with DNA to form adducts with both dG and dA, exogenous exposure favors the formation of only the dG adduct. Lu et al. propose that rapid reaction of exogenous formaldehyde with proteins, followed by selective formation of DPCs at dG bases, may explain the exclusive adduction of dG under their culture conditions. Regardless of the mechanism, the results strongly suggest that levels of N2-methyl-dG provide the most reliable DNA-based biomarker of exogenous formaldehyde exposure.
(DEB-GSH) are more mutagenic than DEB, and expression of GSH transferase enzymes (GSTs) increases DEB mutagenicity in the S. typhimurium tester strain TA1535. These findings led Cho and Guengerich (DOI 10.1021/tx200471x) to investigate the ability of various GSTs to use DEB as a substrate and the formation of DNA adducts by DEB-GSH. Incubation of DEB with GSH and six different GST enzymes (1 rat and 5 human) led to DEB-GSH formation in all cases. The highest enzymatic efficiency was observed with the S,S- stereoisomer, followed by R,R- and meso-, although the differences observed were no more than 3-fold. Using the TA1535 S. typhimurium tester strain, the S,S-DEB-GSH conjugate was the most mutagenic, followed by R,R- and meso-. None of the conjugates was mutagenic in the frameshift TA1537 tester strain. Incubation of DEB-GSH with nucleosides followed by thermal hydrolysis led to six adducts that were structurally characterized. Reaction of DEB-GSH with calf thymus DNA produced the same six adducts, with N6dA-(OH)2butyl-GSH and N7G(OH)2butyl-GSH (see figure) present in the highest amounts. A single intraperitoneal injection of DEB (25 mg/kg) in rats and mice failed to produce DNA adducts in the lung or kidney, but both N6dA-(OH)2butyl-GSH and N7G-(OH)2butyl-GSH were detectable in the liver. Levels (0.19 to 0.27 per 107 bases 6 h after injection) were similar in the two species and decreased somewhat over 48 h. The results demonstrate that GST enzymes efficiently conjugate DEB to produce a product that is more mutagenic than DEB itself. The reaction occurs in vivo and leads to the formation of DNA adducts. This pathway may play an important role in the toxic mechanism of butadiene.
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TRACKING FORMALDEHYDE DNA ADDUCTS
Formaldehyde is a known human and animal carcinogen as a result of its ability to form DNA adducts, protein adducts, and DNA−protein cross-links (DPCs). Exposure to formaldehyde comes from exogenous sources such as vehicle emissions, building materials, and tobacco smoke, and from endogenous sources such as intermediary metabolism and the metabolism of drugs, chemicals, and foods. In vitro, formaldehyde reacts with DNA to form N6-hydroxymethyl-dA, N2-hydroxymethyl-dG, and N4-hydroxymethyl-dC adducts, all of which are unstable 585
dx.doi.org/10.1021/tx3000544 | Chem. Res. Toxicol. 2012, 25, 584−585
