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DCBQ is the major adduct produced from the incubation of pBQ with DNA in vitro. This fact, combined with DCBQ’s relatively simple metabolic disposition suggests that urinary CBQ should be targeted for development as a biomarker of benzene exposure and toxicity.
SPECIAL FEATURES: Iron chelators are a promising new tool in the fight against damage caused by reactive oxygen species that are produced in such conditions as myocardial infarction and neurodegenerative diseases. Kovacevic et al. (DOI: 10.1021/tx100435c) discuss the potential of these compounds with a focus on the article by Hruskova et al. (see Chelators Against Oxidative Stress) that reports a new series of aroylhydrazone chelators.
Chelators Against Oxidative Stress Free iron released during tissue injury can engage in redox cycling reactions, increasing oxidative damage to cells and tissues. Iron chelators such as desferrioxamine, which have been used clinically to treat iron overload conditions, may also be of value to reduce iron-mediated oxidative damage. One promising chelator, salicylaldehyde isonicotinyl hydrazone (SIH) reduces oxidative damage in a number of cell culture models, but its rapid hydrolysis in plasma limits its effectiveness in vivo. Now, Hruskova et al. (DOI: 10.1021/tx100359t) report analogues of SIH with greater stability to hydrolysis that retain their protective efficacy against oxidant damage.
Biomarking Benzene Toxicity Benzene is a bone marrow toxicant and carcinogen that is used industrially and produced in automobile exhaust. Benzene’s toxicity arises, at least in part, from its multistep oxidative metabolism by cytochromes P450 and myeloperoxidases to o-benzoquinone and p-benzoquinone (pBQ). In vitro, pBQ reacts with DNA to form adducts with dC (DCBQ), dA (DABQ), and dG (DGBQ). None of these adducts has been detected in vivo, however, leading Linhart et al. (DOI: 10.1021/tx1003408) to investigate their metabolic fate.
To increase the stability of SIH to hydrolysis, Hruskova et al. replaced the aldimine hydrogen with a methyl or ethyl functionality, yielding HAPI and HPPI, respectively. They also introduced various functional groups into the phenyl ring of HAPI and demonstrated that electron-withdrawing substituents generally led to greater stability. The most stable compound, NHAPI, bearing a 5-nitro phenyl substituent, exhibited 94% recovery after a 180 min incubation with rabbit plasma. All compounds retained the ability to chelate labile iron in H9c2 cardiomyoblast cells. Most compounds significantly reduced the production of reactive oxygen species in response to t-butyl hydroperoxide (t-BHP), and all protected H9c2 cells against t-BHPinduced cell death. Nearly all of the compounds demonstrated reduced short-term (24 h exposure) toxicity compared to SIH in H9c2 cells, but toxicity increased for most upon a 72 h exposure. The analogues demonstrated a range of EC50 values for protection against t-BHP-mediated cellular damage and TC50 values for toxicity at 72 h. Both values correlated with polar surface area and the pKa of the phenolic hydroxyl group. Particularly promising compounds include HAPI, which demonstrated high protective efficacy with low short-term toxicity and NHAPI, characterized by exceptional stability and very low long-term toxicity. Further studies will reveal the therapeutic potential of these new analogues in vivo.
After synthesizing DCBQ, DGBQ, and DABQ, Linhart et al. subjected them to acid hydrolysis to produce the corresponding nucleobases, CBQ, GBQ, and ABQ, respectively. Mild conditions readily yielded CBQ and GBQ from DCBQ and DGBQ, whereas DABQ decomposed to multiple products. This result led the researchers to concentrate their studies on the two former adducts. Following injection of DCBQ or DGBQ into rats, approximately 10% of each compound was recovered unchanged in the urine. When urine from DCBQ-injected rats was treated with mild acid to hydrolyze all glucuronide and sulfate conjugates and convert all nucleosides to nucleobases, 80% of the compound was recovered as CBQ. However, the same procedure using urine from DGBQtreated rats recovered only 15% of the compound as GBQ. Mass spectrometric analysis of untreated urine from DCBQtreated rats revealed the presence of DCBQ-glucuronide and CBQ-sulfate conjugates. In contrast, the urine of DGBQ-treated rats contained a DGBQ-glucuronide, a DGBQ-sulfate, two oxygenated metabolites of DGBQ (DGBQO), a DGBQOglucuronide, and a GBQO-glucuronide. These results explained why acid hydrolysis of urine from DCBQ-treated rats yielded predominantly CBQ, whereas urine from DGBQ-treated rats produced multiple metabolites upon hydrolysis. r 2011 American Chemical Society
Immunosuppressive Lipid Electrophiles Under conditions of oxidative stress, polyunsaturated fatty acids are oxidized to yield diffusible or phospholipid-bound Published: March 21, 2011 283
dx.doi.org/10.1021/tx200052p | Chem. Res. Toxicol. 2011, 24, 283–284
Chemical Research in Toxicology
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reactive electrophiles. The most thoroughly characterized of these is 4-hyroxynonenal (HNE), which forms adducts with proteins and DNA and modulates a number of key cell signaling pathways. To better understand the impact of a wider range of lipid-derived electrophiles, McGrath et al. (DOI: 10.1021/tx100323m) investigated the reactivity, toxicity, and immunosuppressive effects of seven structural analogues of HNE. McGrath et al. synthesized HNE, 4-oxononenal (ONE) and the carboxylic acid metabolites derived by aldehyde oxidation of both compounds, HNEA and ONEA, respectively. They also synthesized 12-carbon analogues bearing a terminal carboxyl group (COOH-HNE and COOH-ONE) and the methyl esters of those compounds (COOMe-HNE and COOMe-ONE). These carboxyl-containing species are similar to electrophiles generated by the hydrolysis of oxidatively damaged phospholipids.
not the only factor. Similarly, the marked reduction in toxicity of COOH-HNE as compared to COOMe-HNE suggests an important role for compound charge, but this observation did not apply to COOH-ONE and COOMe-ONE, which exhibited similar IC50 values. Further work will be required to fully understand the key molecular determinants of toxicity for this class of compounds.
Arsenic-Mediated Carcinogenesis Although chronic exposure to arsenic has been associated with multiple forms of cancer, the exact mechanism of arsenic-mediated carcinogenesis remains unclear. Metabolism of arsenic via biomethylation generates reactive oxygen species (ROS) that can lead to DNA damage. However, arsenic has been shown to transform immortalized normal prostate epithelial (RWPE-1) cells, which are deficient in the biomethylation pathway. This observation led Singh et al. (DOI: 10.1021/tx1003112) to explore potential mechanisms of carcinogenesis in RWPE-1 cells. A 90 day exposure of RWPE-1 cells to sodium arsenite at concentrations from 1 pg/mL to 100 ng/mL resulted in increased cell proliferation. Flow cytometric analysis did not reveal arsenite-dependent alterations in cell cycle progression that readily explained the observed increases in cell numbers, and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated no change in mRNA levels for the cell cycle regulatory proteins Cyclin D1 or p53. In contrast, RT-PCR indicated a striking arsenite-dependent decrease in expression of the mRNA for the pro-apoptotic protein Bax, accompanied by an increase in expression of the mRNA for the antiapoptotic protein Bcl2. These changes suggested that a major contributor to the arsenite-mediated increase in RWPE-1 cell number was suppression of apoptosis. Prior reports had indicated a role for mitochondria in arsenicinduced carcinogenesis. Consistent with this hypothesis, Singh et al. showed that expression of the mRNA for the redox sensitive transcription factor NRF-1 and its target protein mtTFA were elevated in arsenite-treated cells, an observation that was confirmed at the protein level by immunoblot. As a key transcription factor regulating the expression of genes encoded in mitochondrial DNA, increased mtTFA expression indicated that chronic arsenite exposure leads to alterations in mitochondrial function in RWPE-1 cells. An arsenite-mediated increase in mRNA levels for the DNA synthesis/repair protein PCNA, combined with increases in mRNA levels for the nucleotide excision repair (NER) and base excision repair proteins XPC and OGG1, respectively, suggested activation of DNA repair pathways. However, mRNA levels for the NER protein ERCC6 were suppressed, and the comet assay revealed increased levels of nuclear DNA damage in arsenitetreated cells. Furthermore, the PCR amplification and sequencing of specific regions of the mitochondrial genome revealed an arsenite-associated mutation in the mitochondrial ATPase gene. Together, the results indicate that arsenite exposure induces both nuclear and mitochondrial DNA damage, alters mitochondrial function, and suppresses the apoptotic response. All of these findings contribute to our understanding of arsenic-mediated carcinogenesis in the absence of direct ROS generation associated with arsenic biomethylation.
With the exception of HNEA and COOH-HNE, all compounds exhibited similar toxicity in human colon carcinoma cells (RKO) and human monocytic cells (THP-1), with IC 50 values in the range of 16 to 70 μM. In contrast, HNEA and COOH-HNE were minimally toxic, with IC50 values >400 μM. Kinetic evaluation of the reactivity of each compound with Nacetyl-cysteine revealed similar second order rate constants for HNE, COOH-HNE, COOMe-HNE, and ONEA (k = 0.79 1.72/M 3 s), while ONE, COOH-ONE, and COOMe-ONE were approximately 2 orders of magnitude more reactive (k = 170 300/M 3 s), and HNEA did not react. Prior reports had shown that HNE is immunosuppressive in macrophages. McGrath et al. confirmed these findings showing that HNE suppresses bacterial lipopolysaccharide (LPS)- and interferon (IFN)-γ-stimulated cytokine production by THP-1 cells. Of three cytokines tested, interleukin (IL)-1β was the most sensitive to HNE-mediated suppression, followed by IL-6 and tumor necrosis factor (TNF)-R. HNE suppressed cytokine synthesis after a brief 30 min exposure of the cells prior to LPS/IFN-γ addition. However, in the case of IL-6, a 3 h delay between HNE treatment and the addition of stimulus negated the inhibitory effect of HNE. Other lipid electrophiles, including ONE, COOMe-HNE, COOMe-ONE, and ONEA also inhibited LPS/IFN-γ-mediated cytokine synthesis by THP-1 cells. The ineffectiveness of HNEA suggested a correlation between cytokine synthesis inhibition and electrophile reactivity; however, the responses of the cytokines to electrophile exposure were complex, and no consistent correlations with reactivity or toxicity could be made. The low reactivity, toxicity, and immunosuppresive activity of HNEA confirmed the importance of the R,β-unsaturated carbonyl moiety, which is absent in this compound but present in all others. However, the observation that ONE was much more reactive than HNE while having similar toxicity and lower immunosuppressive activity clearly indicates that reactivity is 284
dx.doi.org/10.1021/tx200052p |Chem. Res. Toxicol. 2011, 24, 283–284
