Spotlight pubs.acs.org/crt
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BIOMECHANICAL TOXICITY OF ULTRAVIOLET RADIATION Ultraviolet radiation (UV) from the sun is necessary for the synthesis of vitamin D in the skin, but it is also toxic, causing erythema (redness), photoaging, and cancer. Skin cancer is the most common form of cancer, causing 1.5 million disabilityadjusted life-years and 60,000 premature deaths per year around the world. The mechanisms by which UV causes DNA damage have been extensively studied, but we know little about its effects on the protective, barrier function of the skin. Now, Biniek et al. [(2012) Proc. Natl. Acad. Sci. U.S.A., 109, 17111] address this question. Forming the surface of the epidermis, the stratum corneum (SC) comprises multiple layers of dead cells that contain networks of keratin fibers. The cells are held together by intercellular adhesive structures, the corneodesmosomes, and lipids. Biniek et al. isolated SC from the skin of human cadaver donors and exposed it to different doses of UV. Microtension testing revealed that UV exposure had little effect on stiffness but caused reductions in fracture stress and strain. Bulge testing indicated a decrease in biaxial stiffness and peak stress as a result of UV treatment. Biniek et al. went on to show that UV exposure lowers the energy required to delaminate the layers of the SC and that this effect became more pronounced with increasing depth. Attenuated total reflectance Fourier transform infrared spectroscopy indicated that UV treatment resulted in reduced or modified lipid content, with increased lipid fluidity at the SC surface and decreased fluidity in deeper layers. UV exposure also increased hydration of the SC. The retention of stiffness by UV-treated skin suggests that keratin was not significantly affected. However, the loss of fracture strength and strain combined with the decrease in delamination energy all suggest damage to the corneodesmosomes. These changes are also consistent with alterations in lipid content and composition. The decrease in bulge stiffness can be explained by the changes in lipid composition and the increase in hydration. Biniek et al. developed a mathematical model to relate the effects of UV on the biomechanical properties of the skin. The results show that UV increases the mechanical driving force for cracking while decreasing the skin’s natural ability to resist cracking. They suggest that these are important toxic effects of UV that are not currently considered in the development and evaluation of sunscreens. Carol A. Rouzer
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DISCRIMINATING ARSENATE FROM PHOSPHATE
of the proteins. PBP-2 from Halomonas sp. GFAJ-1 exhibited a 4500-fold discrimination. PBP-2 is highly expressed under conditions of low phosphate and high arsenate to which this bacterium is well adapted. To understand the structural basis of the anion discrimination, Elias et al. obtained a high quality sub-Ångströ m crystal structure of the PBP from P. f luorescens complexed to arsenate. Comparison to comparable structures of the protein− phosphate complex revealed that the two anions are bound in exactly the same mode. Both are held in place by 12 H-bonds, 9 of which are ion−dipole interactions. The difference in As−O versus P−O bond lengths has very little effect on the bond distances or angles of any of these interactions with the exception of one very short distance H-bond between O2 of the anion and the carboxylate of Asp62. In the case of phosphate, this special interaction, classified as a heteromolecular negative-chargeassisted H-bond ((−)CAHB), exhibits nearly canonical bond lengths and angles. In contrast, for arsenate, the geometry is suboptimal. Also notable are very tight van der Waals interactions between each anion and Ala7 and Leu9 of the protein. The steric clash with these residues resulting from arsenate’s larger size is channeled to the CAHB. Mutation of Asp62, Ala7, or Leu9 resulted in a loss of discrimination between arsenate and phosphate while having little effect on phosphate affinity. The results suggest that binding and discrimination have distinct structural determinants. The basis for the extremely high discrimination of Halomonas sp. GFAJ-1 PBP-2 will be the interesting focus of future work. Carol A. Rouzer
Reprinted by permission from Macmillan Publishers Ltd from Elias et al. (2012) Nature, 491, 134.
Copyright 2012. Reprinted by permission from Macmillan Publishers Ltd from Elias et al. (2012) Nature, 491, 134. Copyright 2012.
Phosphate plays multiple critical roles in the biochemistry of life, including energy metabolism, cell signaling, and nucleic acid structure. Thus, the fact that arsenate is almost structurally identical to phosphate in terms of pKa, oxygen atom charge, and thermochemical radius (only 4% larger) poses a dilemma. Failure of some enzymes of energy metabolism to discriminate between the two anions can lead to substitution of arsenate for phosphate and an uncoupling of oxidation from energy storage, a key mechanism of arsenic toxicity. Clearly, a cell’s ability to exclude arsenate is important, leading Elias et al. [(2012) Nature, 491, 134] to explore the mechanism of phosphate/arsenate discrimination by bacterial periplasmic phosphate binding proteins (PBPs). Under phosphate-limited conditions, PBPs bind the anion in the periplasmic space and deliver it to an ATP-dependent transporter for uptake into the cell. Elias et al. cloned, expressed, and purified one PBP each from E. coli, P. f luorescens, and K. variicola, and two PBPs from Halomonas sp. GFAJ-1. Competitive binding studies revealed 500- to 850-fold greater binding affinity for phosphate over arsenate in the case of 4 © 2012 American Chemical Society
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Chemical Research in Toxicology
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Spotlight
NANOTOXICITY ACROSS THE GENOME
membrane-related genes, while most other pathways led to effects at early time points. The results revealed unique mechanisms or patterns of mechanisms for each material that were only detected at one time point, confirming the value of this genome-wide, comprehensive approach for the assessment of nanoparticle toxicity. Carol A. Rouzer
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ROLE FOR TRNA MODIFICATION AFTER DNA DAMAGE In Saccharomyces cerevisiae, the enzyme tRNA methyltransferase 9 (Trm9) catalyzes the methylation of uridines in the wobble position of tRNA codons to facilitate the synthesis of 5-methoxycarbonylmethyluridine (mcm5U). The substrate tRNAs code for Arg, Lys, Glu, or Gln. The presence of mcm5U enhances binding to codons ending in A for Arg and Glu residues, and speeds translation. Cells deficient in Trm9 (trm9Δ) are more sensitive to killing by ionizing radiation, hydroxyurea, and alkylating agents than wild-type (WT) cells, suggesting a role for Trm9-mediated translation regulation in the DNA damage response. One possible mechanism by which this may occur is through regulation of translation of the Rnr1 and/or Rnr3 subunits of the ribonucleotide reductase (RNR) complex, a key enzyme in the biosynthesis of dNTPs. RNR activity markedly increases during DNA synthesis and after DNA damage in order to provide adequate dNTPs for repair. Prior studies show decreased levels of Rnr1 and Rnr3 in trm9Δ cells, which cannot be explained by reduced transcription of their respective genes. This led Patil et al. [(2012) Cell Cycle, 11, 3656] to investigate the role of Trm9 in RNR regulation in greater detail. When cells were synchronized in various stages of the cell cycle, trm9Δ cells exhibited lower levels of Rnr1 protein than WT cells. Analysis by chromatography-coupled MS revealed differences in levels of specific tRNA modifications in S-phase synchronized versus asynchronous cells, including an ∼2-fold increase in the level of mcm5U in S-phase. The results suggested a role for Trm9 in Rnr1 translation. After treatment with the DNA alkylating agent methyl methanesulfonate (MMS), trm9Δ cells exhibited a delayed progression to S-phase relative to WT cells. This delay was alleviated by re-expressing TRM9, overexpressing RNR1, or deleting the gene for the RNR inhibitor Sml1 in trm9Δ cells. A luciferase reporter construct designed to measure translation of transcripts containing a 12 base pair sequence of mcm5Udependent codons demonstrated decreased translation of these codons in trm9Δ cells as compared to WT cells. This could explain low levels of Rnr1 in trm9Δ cells since the transcript of RNR1 shows biased use of the mcm5U-dependent codons AGA for Arg and GAA for Glu. To test this hypothesis, Patil et al. replaced the RNR1 in WT and trm9Δ cells with opt-RNR1, a gene using all high-usage codons optimized for rapid, mcm5Uindependent translation. Expression of opt-RNR1 resulted in higher Rnr1 protein levels and a more rapid transition to S-phase following MMS treatment in both WT and trm9Δ cells. These results indicate that Rnr1 protein levels are rate-limiting for the S-phase transition following DNA damage and provide further support for the hypothesis that selective translation of RNR1 transcripts is regulated by Trm9-dependent tRNA modifications during the DNA damage response. Carol A. Rouzer
Reprinted from Reyes et al. (2012) ACS Nano, published online Oct 6, DOI: 10.1021/nn302815w.
Copyright American Chemical Society. published online Reprinted from Reyes et 2012 al. (2012) ACS Nano, Oct 6, DOI: 10.1021/nn302815w. Copyright 2012 American Chemical Society.
Engineered zinc nanomaterials are among the most widely used nanoparticles, with current applications in cosmetics, paints, plastics, sunscreens, and tires. These are also among the most toxic nanoparticles, with adverse effects observed in a broad range of species. Current methods of testing fail to provide a comprehensive assessment of all possible toxic mechanisms. Some studies indicate a major role for Zn2+ ion resulting from nanoparticle dissolution, while others suggest particle-specific mechanisms. To address these questions, Reyes et al. [(2012) ACS Nano, published online Oct. 6, DOI: 10.1021/nn302815w] employed a genome-wide high-throughput approach to evaluate the toxicity of zerovalent zinc and zinc oxide nanoparticles (nZn and nZnO, respectively) and ZnCl2 as a source of Zn2+ ions. Reyes et al. used an E. coli library comprising 4000 clones, each representing the knockout of a unique single gene in the bacterial genome. They exposed all clones to each material at the concentration that caused 50% inhibition of growth of wild-type bacteria (IC50) and monitored growth at four time points representing lag phase, exponential, and stationary growth. The results identified 173 clones that exhibited increased sensitivity to at least one of the materials at a minimum of one time point. These were subjected to two further testing events to elucidate the IC50 of all three materials at two separate time points. Of the 173 clones, 112, 77, and 81 were sensitive to ZnCl2, nZn, and nZnO, respectively, with 70, 17, and 19 respective clones being unique for each of the materials and 24 clones sensitive to all three. Cluster analysis indicated distinct groups of clones that were sensitive at early (106 clones) versus late (91 clones) time points (24 shared clones) and distinguished between groups of high (26 clones), intermediate (94 clones), and mild (53 clones) sensitivity. In general, IC50 values of the sensitive clones increased over time, though this trend was not found in the high sensitivity clones, which tended to be late or shared time point responders. Functional annotation clustering of the sensitive clones revealed 8 clusters in 5 general biological categories. These included metal homeostasis (transport and binding), central metabolism (monosaccharide synthesis), regulation and signaling (transcription and two component signaling), general transport, and membrane-related (lipopolysaccharide synthesis and membrane structure) categories. Clones bearing mutations in Zn metabolism exhibited the lowest IC50’s at all time points. In general, persistent toxicity was associated with mutations in 2626
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