In This Issue pubs.acs.org/crt
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SPECIAL FEATURES As a perfect follow up to the recent review by Fukuto et al. (DOI 10.1021/tx2005234) on the basic chemistry and biochemistry of small signaling gaseous molecules, Toledo and Augusto (DOI 10.1021/tx300042g) now provide an in depth analysis of the interaction of nitric oxide with its protein targets. This comprehensive review includes consideration of the kinetics and mechanisms of nitric oxide’s reactions in the cellular context, and relates them to the physiological and pathophysiological roles of this important molecular signal.
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SUBTLE DANGERS OF NANOPARTICLES
(200 mg/L) or Cu ions (2 mg/L), suppressed the expression of pvdS, the gene encoding a sigma factor that regulates the expression of PVD-related genes. Similarly, CuO NPs had no or only modest effects on the expression of two genes involved in ferribactin synthesis. However, expression of the pvdE gene, responsible for transport of ferribactin across the inner membrane to the periplasmic space, was inhibited by both Fe ions and CuO NPs, but not Cu ions. The same pattern of suppression applied to genes involved with PVD maturation in the periplasm, pvdQ, pvdN, and pvdO, and PVD export and recycling, pvdR, pvdT, and opmQ. For all of these genes, expression was suppressed by Fe ions and CuO NPs, but not Cu ions. In contrast, the expression of only one of three genes potentially associated with uptake of Fe-bound PVD into the cell was suppressed by CuO NPs. Dimkpa et al. concluded that the effect of sublethal CuO NP exposure on PcO6 PVD metabolism is a CuO NP-specific phenomenon since bulk CuO and Cu ions did not suppress PVD levels, and ZnO NPs actually caused an increase. Since PcO6 and similar bacteria play an important role in promoting plant root growth and the resistance of plants to various stressors, the subtle effects of sublethal concentrations of NPs should not be overlooked.
As the use of nanomaterials continues to grow, the risk of environmental contamination by nanoparticles (NPs) increases. This raises concern with regard to the toxicity of NPs to humans as well as to other species. Of particular interest to Dimkpa et al. (DOI 10.1021/tx3000285) is the toxicity of NPs to beneficial soil bacteria that are involved in bioremediation, element cycling, and plant growth promotion. To address this concern, they investigated the effects of CuO NP exposure on the root colonizing bacterium Pseudomonas chlororaphis O6 (PcO6). To enable them to compete and survive in iron-deficient environments, bacteria often produce iron-binding siderophores including the fluorescent pyoverdines (PVDs), which are synthesized by many pseudomonads. PVDs consists of a nonribosomal peptide, ferribactin, and a dihydroxiquinoline chromophore. The bacteria export PVD into the environment where it binds iron ions for transport into the cell. Dimkpa et al. showed that, while high concentrations of CuO NPs are toxic to PcO6, sublethal concentrations selectively decrease the levels of PVD produced by the cells. In contrast, neither bulk CuO nor exposure to Cu ions at concentrations comparable to levels released by CuO NPs had any effect on PVD levels produced by the bacteria. To understand the effect of nonlethal CuO NP exposure on PcO6 PVD levels, Dimkpa et al. used sequence homology with the well-characterized Pseudomonas aeruginosa genome to identify PcO6 genes involved in PVD synthesis, processing, and transport. Then, RT-PCR revealed changes in the expression of those genes in response to CuO NP exposure. The results showed that iron ions (100 μM), but not CuO NPs © 2012 American Chemical Society
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ACTIVATION OF A MUTAGENIC ALKALOID
The alkaloid ellipticine from Apocynaceae species of plants possesses promising antitumor and antiviral activities. A potent mutagen, ellipticine is a DNA intercalator and topoisomerase II inhibitor. It also forms DNA adducts following activation by cytochromes P450 1A1, 1B1, 3A1, and 3A4. Of these, the 3A family P450s demonstrate the greatest ability to convert ellipticine to its 12-hydroxy and 13-hydroxy metabolites. These are precursors of the highly reactive ellipticine-12ylium and -13-ylium, respectively, which form adducts with Published: May 21, 2012 971
dx.doi.org/10.1021/tx3001384 | Chem. Res. Toxicol. 2012, 25, 971−972
Chemical Research in Toxicology
In This Issue
Exposure of ovarian carcinoma A2780 cells to 1 in the presence of UVA led to platination of nuclear DNA at a level 16-fold higher than was observed from exposure to the same concentration of cisplatin. The level of DNA platination correlated with cytotoxicity. Incubation of calf thymus DNA (ctDNA) with 1 in vitro effected a concentration- and light exposure-dependent platination of the DNA that was more rapid and extensive than that observed with cisplatin. Visible light could also photoactivate 1, although it was less effective than UVA. No 1-dependent DNA platination occurred in the absence of light. Use of thiourea to labilize monofunctional adducts revealed that >65% of the adducts of 1 with DNA were bifunctional. Gel electrophoresis of a linearized, radiolabeled plasmid treated with photoactivated 1 indicated that approximately 12% of the adducts were in the form of interstrand cross-links. These results suggest that at least 50% of the adducts of 1 with DNA were intrastrand cross-links. Treatment of plasmid pUC19 with photoactivated 1 resulted in relaxation of supercoiling. Native gel electrophoresis of the modified plasmids revealed a relaxation angle of 28°, a value larger than that seen with cisplatin (13°) or its clinically ineffective isomer transplatin (9°). Compound 1 was more effective than cisplatin or transplatin in its ability to decrease the fluorescence of ethidium bromide-treated DNA or increase the fluorescence of terbium ion-treated DNA. These results suggest that 1 disrupts the DNA helical conformation more profoundly than either cisplatin or transplatin. Treatment with photoactivated 1 blocked T7 RNA polymerase-dependent transcription of the pSP73kB plasmid, leading to a greater variety of fragments than were obtained with cisplatin treatment. Photoactivated 1 was more effective than cisplatin in blocking RNA polymerase II-dependent transcription of pCMV-Gluc. A synthetic oligonucleotide containing a single intrastrand cross-link of 1 exhibited a complete block of T7 RNA polymerase-dependent transcription, while a comparable cross-link of cisplatin effected a 73% block. Together, the results suggest that photoactivated 1 is a more efficient DNA cross-linking agent than cisplatin; however, due to its lack of toxicity in the absence of light, 1 may be a safer antitumor agent for clinical use.
guanine bases of DNA. Now, Stiborová et al. (DOI 10.1021/ tx3000335) report on conditions that increase P450-mediated ellipticine-DNA adduct formation. Incubation of ellipticine with P450 3A4, along with NADPH:P450 reductase and NADPH led to the formation of 5 metabolites, with 12-, 13-, and 9-hydroxyellipticine predominating (see figure). Inclusion of cytochrome b5 in the reaction mixture resulted in a 1.6- and 2.7-fold increase in the formation of 12- and 13-hydroxyellipticine, respectively, but not in the detoxication product 9-hydroxyellipticine. Addition of cytochrome b5 to incubations of calf thymus DNA, P450 3A4, NADPH:P450 reductase, and NADPH produced comparable respective increases in levels of the DNA adducts formed from 12- and 13-hydroxyellipticine. In contrast, addition of apo-cytochrome b5 or cytochrome b5 reconstituted with Mn-protoporphyrin IX had no effect on P450 3A4mediated ellipticine metabolism. The ratio of 13-hydroxyellipticine to 12-hydroxyellipticine produced by P450 3A4-mediated metabolism of ellipticine was 1.3:1; however, DNA adduct formation from 13-hydroxyellipticine occurred at 7-fold higher levels than that from 12hydroxyellipticine. Addition of N,O-acetyltransferases (NAT) or sulfotransferases (SULT) along with their respective cofactors to incubations of 13-hydroxyellipticine and DNA substantially increased DNA adduct formation. The most active SULT, SULT1A3, effected a 68.5-fold increase in DNA adduct levels, while the most active NAT, NAT2, increased DNA adduct formation 188-fold. Molecular modeling of three different pathways of heterolytic dissociation of the 12- and 13-hydroxyellipticines to the corresponding yliums supported a proton-mediated mechanism. Furthermore, dissociation of 13-hydroxyellipticine is energetically favored compared to 12-hydroxyellipticine, and dissociation of the sulfate and acetyl esters are favored over the free alcohols. Thus, the modeling results supported the experimental observations. Stiborová et al. concluded that P450 complex formation with cytochrome b5 and conjugating enzymes may play an important role in the activation and mutagenicity of ellipticine.
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PHOTOACTIVATION FOR ANTITUMOR TOXICITY
Cisplatin (cis-[PtCl2(NH3)2] is a widely used antitumor agent that acts primarily by adducting DNA to form intrastrand crosslinks. Toxicity to the patient limits the use of this highly effective drug, indicating the need for a more selective alternative. One approach is the design of photoactivatable Pt(IV) diazido complexes, such as trans,trans,trans-[Pt(N3)2(OH)2(pyridine)2] (1). This compound is nontoxic in the absence of light but upon exposure to ultraviolet or visible light is converted to a highly reactive Pt(II) analogue. Now, Pracharova et al. (DOI 10.1021/tx300057y) report on the interaction of 1 with DNA. 972
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