In This Issue pubs.acs.org/crt
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SPECIAL FEATURES Over the past 25 years, we have amassed large quantities of data on the role of metabolizing enzymes in chemical carcinogenesis. Although some of these enzymes catalyze detoxication, much greater interest has focused on the role of metabolism in the activation of pro-carcinogens. Now, Rendic and Guengerich (DOI 10.1021/tx300132k) provide a comprehensive review of what we know about the bioactivation and detoxication of carcinogenic compounds. The ToxCast (“Toxicity foreCaster”) program is the U.S. EPA’s response to the need for rapid, efficient, and accurate in vitro assessments of toxicity so that the safety of the huge number of new compounds being generated annually can be evaluated. As phase I of the ToxCast program ends, Kavlok et al. (DOI 10.1021/tx3000939) summarize its successes, challenges, and plans for the future. The toxicity of benzene is focused primarily on the hematopoietic system. To better understand benzene’s leukemogenicity and hematotoxicity, Wang et al. (DOI 10.1021/tx3001169) evaluate its effects and those of its metabolites on bone marrow stem cell populations.
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STRESS-INDUCING COBALT NANOPARTICLES
p53 and Rad51 protein levels. A selective ATM inhibitor suppressed the accumulation of p53 and Rad51, and the phosphorylation of p53, confirming that these responses were ATM-dependent. Treatment of A549 cells with Nano-TiO2, a nontoxic preparation of nanoparticles of size and surface area similar to those of Nano-Co, failed to elicit any of these cellular responses. The finding that pretreatment of A549 cells with the antioxidants N-acetyl-cysteine or catalase reduced Nano-Coinduced DNA damage and the DNA damage response confirmed that these phenomena were mediated by ROS produced in response to Nano-Co. Wan et al. concluded that oxidative stress plays a major role in the toxic response to Nano-Co.
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As the industrial and biomedical applications of cobaltcontaining nanoparticles continue to expand, their potential toxicity is a focus of increasing concern. This led Wan et al. (DOI 10.1021/tx200513t) to conduct a detailed exploration of the mechanism of toxicity of Nano-Co, a preparation of 20 nm particles comprising ∼90% Co and ∼10% Co3O4. Exposure of human A549 lung epithelial cells to Nano-Co resulted in a concentration-dependent uptake of the particles. Nano-Co caused a significant reduction in A549 cell viability at concentrations of 20 μg/mL or higher, so Wan et al. restricted their studies to concentrations below this threshold. Even at these lower concentrations, Nano-Co induced reactive oxygen species (ROS) production in A549 cells as detected by 2′,7′dichlorodihydrofluorescein diacetate fluorescence and increased levels of the DNA oxidation product 8-hydroxy-2′-deoxyguanosine (8-OH-dG). The comet assay confirmed that Nano-Co induced DNA fragmentation in the cells. DNA double strand breaks induce the phosphorylation of the protein kinase ataxia telangiectasia mutated (ATM), which in turn, phosphorylates the histone H2AX and promotes the local accumulation of the DNA damage repair protein Rad51. ATM activation also promotes the accumulation and phosphorylation of the DNA damage response protein p53. Accordingly, NanoCo exposure resulted in the appearance of phosphorylated H2AX foci in the nucleus of A549 cells as detected by immunofluorescence microscopy. Western blot analysis revealed a time- and concentration-dependent increase in the phosphorylation of ATM, H2AX, and p53, and an increase in © 2012 American Chemical Society
FLAME RETARDANTS: FRIEND OR FOE?
The widespread use of polybrominated compounds as flame retardants is due to their ability to generate halogen radicals that quench the free radicals necessary to propagate fire. Published: July 16, 2012 1283
dx.doi.org/10.1021/tx3002498 | Chem. Res. Toxicol. 2012, 25, 1283−1284
Chemical Research in Toxicology
In This Issue
Consequently, estimated human exposure to HMF is as high as 30 mg/day. Studies in rats indicate that HMF may be a carcinogen; however, it fails to exhibit significant genotoxicity in conventional assays. One possible mechanism of HMFmediated carcinogenicity is based on its bioactivation by sulfotransferases (SULT), which convert HMF to the reactive electrophile, 5-sulfooxymethylfurfural (SMF, see figure). In the presence of SULTs, HMF is mutagenic to the Salmonella typhimurium TA100 tester strain. To further test this hypothesis, Monien et al. (DOI 10.1021/tx300150n) studied the mutagenicity and DNA reactivity of HMF in the V79 Chinese hamster lung fibroblast cell line and in V79 cells expressing human SULT1A1 (V79-hSULT1A1). To measure mutagenicity, Monien et al. studied the ability of the cell lines to survive in the presence of 6-thioguanine (6TG). 6TG is a toxic guanine analogue that requires activation by the enzyme, hypoxanthine−guanine phosphoribosyltransferase (HPRT). Mutations in the hprt gene allow cells to survive in the presence of 6-TG. Incubation of V79-SULT1A1 cells with HMF resulted in a concentration-dependent mutation of hprt that was not observed in the V79 control cells. Exposure of DNA to SMF in vitro followed by hydrolysis and MS analysis revealed four adducts identified on the basis of the loss of the 5-methylfurfural cation (m/z = 109). The MS data indicated the presence of one adduct each to dAdo and dGuo, and two adducts to dCyt. Incubation of the individual 2′deoxyribonucleosides with SMF yielded the same adducts. Comparison of the dAdo and dGuo adducts to synthetic standards of N6-((2-formylfuran-5-yl)methyl)-2′-deoxyadenosine (N6-FFM-dAdo) and N2-((2-formylfuran-5-yl)-2′-deoxyguanosine (N2-FFM-dGuo) by MS and 1H NMR confirmed the identities of these two adducts and provided the needed tools to develop an LC-MS assay for their quantification. Because the aldehyde group of HMF can form an imine with the exocyclic amines of dAdo and dGuo, Monien et al. incubated these 2′-deoxyribonucleosides with HMF followed by NaBH3CN to stabilize the imine bond. MS analysis confirmed the formation of adducts at the N6-position of dAdo and the N2-position of dGuo. However, incubation of HMF with intact DNA did not yield these products except at very high concentrations of the compound. Exposure of V79-SULT1A1 cells to HMF followed by isolation and analysis of cellular DNA revealed concentrationdependent formation of N6-FFM-dAdo and N2-FFM-dGuo at levels reaching 0.79 adducts/108 nucleosides and 3.3 adducts/ 108 nucleosides, respectively, at an HMF concentration of 2.5 mM. No adducts were formed during exposure of V79 control cells to HMF. Incubation of V79 cells with SMF (0.3 mM) resulted in 0.46 N6-FFM-dAdo/108 nucleosides and 4.0 N2FFM-dGuo/108 nucleosides. Subsequent incubation of the cells for up to 24 h indicated that these adducts are highly stable to DNA repair. Monien et al. concluded that HMF is an efficient substrate for hSULT1A1, which activates the compound to the reactive electrophile SMF. Subsequent DNA adduction by SMF could be a mechanism for the rodent carcinogenicity of HMF.
However, polyhalogenated compounds are often prone to bioaccumulation. This led to the phasing out of the polybrominated diphenyl ether (PBDE) flame retardants, which had been heavily used until they were designated as persistent organic pollutants according to the Stockholm Convention. Today, several different chemicals have replaced PBDEs, including 2-ethylhexyl tetrabromobenzoate (TBB) and bis(2-ethylhexyl) tetrabromophthalate (TBPH). Unfortunately, little is known about the metabolism or toxicity of these compounds, despite the fact that their prevalent use in consumer products makes the possibility of human exposure high. The nonbrominated analogue of TBB, 2-ethyhexyl benzoate, is a nontoxic food additive, while the nonbrominated analogue of TBPH, bis(2-ethyhexyl)phthalate, is a known toxicant that is metabolically activated by esterase-mediated cleavage of one of the two ester bonds. To better understand the potential toxicity of TBB and TBPH, Roberts et al. (DOI 10.1021/tx300086x) investigated their metabolism. Both human and rat liver microsomes rapidly metabolized TBB. MS analysis of the incubation mixtures following reaction with human liver microsomes (HLM) and treatment with diazomethane identified the methyl ester of tetrabromobenzoic acid (TBBA), suggesting that TBBA was the major metabolite. Comparison of the chromatographic behavior and mass spectrum with that of a synthetic standard confirmed the compound identification. Microsomal metabolism of TBB did not require NADPH, indicating that cytochromes P450 were not involved. The rapid conversion of TBB to TBBA by porcine carboxylesterase (PCE) suggested that the responsible metabolic enzyme(s) was likely a tissue esterase. Both rat and human liver microsomes and cytosol in addition to rat and human intestinal microsomes efficiently converted TBB to TBBA. In contrast, incubation of TBPH with HLM resulted in no decrease in compound concentration, and no metabolites were detected. PCE did hydrolyze the ester bond of TBPH, but at only 1% of the rate of hydrolysis of its nonbrominated analogue. In addition, Roberts et al. were unable to demonstrate conjugation of the metabolites. The investigators concluded that the metabolism of TBB to TBBA may reduce the potential for bioaccumulation, while simultaneously raising the concern for TBBA’s potential toxicity. The absence of metabolism of TBPH may increase its bioaccumulation potential and could explain the presence of much higher concentrations of TBPH relative to TBB in marine mammals.
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METABOLIC ACTIVATION OF A COMMON CARCINOGEN
5-Hydroxymethylfurfural (HMF) is a Maillard reaction product that is present in foods, beverages, and cigarette smoke. 1284
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