SPOTLIGHT pubs.acs.org/crt
GENOTOXICITY OF HYDROXYUREA Copy number variants (CNVs) are deletions or duplications in genomic DNA that range from 50 to more than a million base pairs in length. Although CNVs are distributed throughout the genome of healthy individuals, they frequently arise de novo as new mutations and can cause a number of genetic and developmental disorders. Recurrent CNVs, observed at regions flanked by large segmental duplications, are believed to result from nonallelic homologous recombination during meiosis, whereas nonrecurrent CNVs are not associated with regions of sequence duplication. The mechanism of formation of nonrecurrent CNVs is not well understood. Their recent discovery that the DNA polymerase inhibitor aphicolin (APH) induces CNVs led Arlt et al. [(2011) Proc. Natl. Acad. Sci. U.S.A., 108, 17360] to hypothesize that nonrecurrent CNV formation could be associated with replicative stress. They tested this hypothesis by investigating the effects of hydroxyurea (HU), an inhibitor of ribonucleotide reductase, on CNV formation. HU induces replicative stress through perturbation of nucleotide pools that leads to stalling of the replication fork. Treatment of immortalized human fibroblasts with HU or APH for 72 h led to similar increased frequencies of CNVs. HU slowed the cell doubling time and reduced colony forming ability, possibly resulting in an underestimation of CNV induction. HU induced both deletions and duplications, and the size of HU-induced CNVs (1 kb to 35.7 Mb) was similar to those observed in APH-treated as well as in untreated cells. HU-induced CNVs were distributed throughout the genome, but some striking hotspots were observed, particularly at 3q13.31 at the LSAMP gene and 7q11.2 at the AUTS2 gene. These regions were also hotspots for APH-induced and spontaneous CNVs. Sequence analysis of the breakpoint junctions of HU-induced CNVs revealed short regions of microhomology, blunt ends, and short insertions that were, in some cases, combined into a single complex CNV. These findings revealed a structure similar to those of spontaneous nonrecurrent human CNVs, suggesting a common mechanism for their formation. The results suggest that nonrecurrent CNVs result from multiple mechanisms associated with replicative stress. They also suggest a previously unrecognized genotoxicity for HU, which is commonly used to treat sickle cell anemia, some cancers, and HIV infection. Further studies to determine if HU exposure has similar effects in the germline in vivo are needed. Carol A. Rouzer
’ PREDICTING AROMATIC AMINE MUTAGENICITY
Reprinted from Shamovsky et al. (2011) J. Am. Chem. Soc., 133, 16168. Copyright 201l American Chemical Society. Aromatic amines and heterocyclic aromatic amines (Ar-NH2) have many potential industrial and pharmaceutic applications. However, concern regarding the mutagenicity and carcinogenicity of some ArNH2’s has limited their use. The toxicity of mutagenic Ar-NH2’s results primarily from their cytochrome P450-mediated metabolism to a hydroxylamine, followed by hydrolytic dissociation to a reactive nitrenium ion. Understanding the mechanistic foundation for the susceptibility to these reactions would allow an accurate prediction of mutagenic potential. To achieve this goal, Shamovsky et al. [(2011) J. Am. Chem. Soc., 133, 16168] combined quantum mechanical calculations with structure activity relationship studies to determine the mechanism of P450 1A2-mediated activation of a series of p-substituted anilines and 2-aminopyridines. The binding site for Ar-NH2’s in P450 1A2 is planar and has a narrow elongated shape that favors the binding of p-substituted rings. Hydrogen bonding between nearby threonine-124 and the pyridine nitrogen stabilizes the binding of 2-aminopyridines. r 2011 American Chemical Society
Density functional theory calculations revealed that Ar-NH2’s optimally bound in the P450 1A2 active site were too far away to react with the oxygen atom of the Compound I oxenoid intermediate, which is considered the most important reactive form of the enzyme. Shamovsky et al. concluded that the dianionic ferric-peroxo intermediate (Compound 0) catalyzes Ar-NH2 hydroxylation (see Figure). Their reaction mechanism begins with a rate-limiting deprotonation of the amino group of the Ar-NH2, yielding AR-NH . The next step requires protonation of the proximal oxygen of Compound 0. The result is weakening of the O O and O H bonds to heterolytic cleavage. Cleavage of the O O bond yields OH+, which reacts with ARNH via an SN2 mechanism to form the product hydroxylamine (Ar-NHOH). Cleavage of the O H bond regenerates enzyme bound Ar-NH2 and produces a hydroperoxyl radical. The net effect of this reaction is the Ar-NH2-catalyzed generation of reactive oxygen species. For most substrates, the energetics of the reaction favor the formation of Ar-NHOH. This reaction mechanism predicts that the mutagenicity of ArNH2’s will be favored by molecular structures that stabilize binding in the P450 1A2 active site, substituents that stabilize the Ar-NH intermediate, and factors that favor subsequent proton-assisted heterolytic dissociation of the N O bond of ArNHOH to form the nitrenium ion. These conclusions, which are consistent with what is currently known about Ar-NH2 mutagenicity, provide a framework to predict which of these potentially useful compounds is likely to be toxic. Carol A. Rouzer
Published: December 19, 2011 2059
dx.doi.org/10.1021/tx200484k | Chem. Res. Toxicol. 2011, 24, 2059–2060
Chemical Research in Toxicology
’ ENDOGENOUS AHR-MEDIATED TUMOR PROMOTION
Reprinted by permission from Macmillan Publishers Ltd. from Opitz et al. (2011) Nature, 478, 197. Copyright 2011. The aryl hydrocarbon receptor (AHR) is well-known to toxicologists for its role in controlling xenobiotic metabolism. Activation of the AHR by compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin leads to the induction of cytochrome P450 isoforms and other enzymes that detoxicate and in some cases activate the xenobiotic compound. However, growing evidence suggests the presence of endogenous ligands that modulate AHR activity. Now, Opitz et al. [(2011) Nature, 478, 197] report that kynurenine (Kyr) a metabolite of tryptophan (Trp) is an endogenous AHR ligand that promotes tumor growth. A screen of human tumor cell lines revealed degradation of Trp and high levels of Kyn in glioma cells. Use of selective inhibitors and shRNA knockdown indicated that the Trp metabolism observed in these cells was attributable to the enzyme tryptophan-2,3-dioxygenase (TDO). TDO protein levels correlated with malignancy and proliferation index in human brain tumor specimens as well as other cancer types. Kyn suppressed the proliferation of allogeneic T-cells in coculture with TDO-expressing glioma cells, and tumor TDO protein levels correlated with reduced antitumor immune responses to glioma xenografts in mice. In addition, TDO expression increased glioma cell clonogenicity and migration in culture and promoted the growth of glioma xenografts in vivo. These findings suggest that Kyn acts as a tumor promotor directly by stimulating tumor growth and indirectly by inhibiting antitumor immunity. Kyn treatment of glioma cells resulted in a strong induction of AHR responsive genes. Kyn bound directly to the AHR with a KD of 4 μM and induced AHR activity in a luciferase expression assay with an EC50 of 36.6 μM. Knockdown of Tdo resulted in decreased AHRregulated gene expression in glioma cells. Ahr-deficient mice mounted a stronger immune response to Tdo-expressing glioma xenografts than did Ahr-proficient mice.
SPOTLIGHT
Tdo-expressing tumors grew faster than Tdo-deficient tumors, but the effect was much greater in Ahr-proficient than Ahr-deficient hosts. These results suggest that the effects of Kyn on tumor growth results from both autocrine regulation of tumor AHRdependent genes and paracrine regulation of host AHR-dependent genes. The finding that TDO protein expression correlates with AHR target gene expression in human glioma, B-cell lymphoma, Ewing sarcoma, and carcinoma of the bladder, cervix, colon, lung, and ovary suggests that TDO-mediated Kyn formation is an important tumor-promoting mechanism in multiple human cancers. Carol A. Rouzer
’ PROTECTION AGAINST OXIDATIVE STRESS Oxidative stress plays a role in the inflammatory response to many toxicants. Reactive oxygen species generated during oxidative stress damage endogenous molecules, yielding oxidation-specific epitopes (OSEs). OSEs are recognized as danger signals by innate immune receptors and trigger an inflammatory response. Malondialdehye (MDA), a major product of lipid peroxidation, forms protein adducts that are recognized as OSEs. Accumulation of MDA adducts in lesions such as atherosclerotic plaques and the retina of patients suffering from age-related macular degeneration (MDA) led Weismann et al. [(2011) Nature, 478, 76] to explore its role in these diseases. Weismann et al. used affinity beads coated with malondialdehydeacetaldehyde- (MAA)-modified polylysine to isolate MDA-binding proteins in mouse plasma. MAA is a complex adduct formed from two molecules of MDA and one of acetaldehyde derived from MDA. The major protein isolated in this way was complement factor H (CFH), a regulator of the pro-inflammatory complement system. Competitive ELISA assays confirmed direct binding of CFH to MDA- and MAA-modified proteins, and showed that the interaction was specific for these and not other oxidation-derived modifications. The structure of CFH comprises 20 globular short consensus repeats (SCRs). Weismann et al. showed that the CFH binding site was localized to SCR7 and SCR20. A variant with histidine at position 402 in SCR7, H402, exhibited markedly reduced binding affinity for MDA-modified proteins when compared to the more common variant carrying tyrosine at that position, Y402. This helps to explain why the H402 variant is a significant risk factor for AMD and supports the importance of CFH binding to MDA modifications in preventing this disease. Immunohistochemistry revealed MDA as well as CFH in the retinas from patients with AMD. MDA was also present on the surface of necrotic retinal pigment epithelial cells, where it colocalized with CFH. Analysis of atherosclerotic plaques confirmed, again, the presence of MDA and CFH in overlapping lesion areas. CFH acts as a cofactor for serine protease factor I, which degrades the pro-inflammatory complement factor C3b into the inactive fragment iC3b. Weismann et al. showed that binding of CFH promotes the conversion of C3b to iC3b on MDA-decorated surfaces, thus protecting against the inflammatory activation of complement. They further showed that CFH blocked the secretion of the proinflammatory cytokine interleukin-8 (IL-8) from macrophages stimulated with MAA-modified bovine serum albumin (BSA) and the formation of KC (the murine equivalent of IL-8) in the retinas of mice injected intravitreally with MAA-modified BSA. Together, the results confirm that MDA-protein modifications serve as OSEs and demonstrate that recognition of these epitopes by CFH helps to protect against an excessive inflammatory response resulting from MDA accumulation. Carol A. Rouzer 2060
dx.doi.org/10.1021/tx200484k |Chem. Res. Toxicol. 2011, 24, 2059–2060
