Spotlight Mito-Toxic SPANs Snake presynaptic phospholipase A2 neurotoxins (SPANs) are a major toxic component of many snake venoms. These enzymes attack the terminals at the neuromuscular junction, inducing paralysis. SPANexposed nerve terminals exhibit swelling, increases in cytosolic Ca2+, and mitochondrial rounding and depolarization. These same changes can be induced by exposing neurons to the fatty acid and lysolipid products of SPAN hydrolysis. Now, Rigoni et al. [(2008) J. Biol. Chem., published online Sept. 22, DOI: 10.1074/jbc.M803243200] have explored the interaction of SPANs with nerve cells in greater detail by using four different SPANs labeled with Alexa568 or fluoresceine. Fluorescence microscopy allowed the investigators to monitor the rapid uptake of the SPANs into spinal cord motor neurons or cerebellar granular neurons. The SPANs then localized
Diclofenac + Bacteria ) Toxic Diclofenac (DCLF) is a widely prescribed nonsteroidal antiinflammatory drug that can trigger idiosyncratic hepatotoxic drug reactions. The rarity of these reactions and the lack of an appropriate animal model have made it difficult to determine their mechanism. Now, Deng et al. [(2008) J. Pharmacol. Exp. Ther., published online Sept. 18, DOI: 10.1124/jpet.108.140335] have begun to address this issue based on the observation that bacterial lipopolysaccharide (LPS) exposure enhances and antibiotic treatment protects against DCLF toxicity. Deng et al. proposed that DCLF causes intestinal ulceration, leading to a release of bacteria or LPS into the bloodstream, and that the resulting inflammatory stress exacerbates DCLF-mediated liver damage. To support this hypothesis, they used K-means clustering analysis to show a similarity between the gene expression pattern of the livers of rats treated with LPS and those treated with a toxic dose of DCLF. Rats treated with antibiotics sufficient to sterilize the gut and then administered a hepatotoxic dose of DCLF exhibited markedly reduced blood alanine aminotransferase levels and histochemical evidence of liver damage than rats treated with DCLF alone. Hierarchical clustering analysis of gene expression profiles from the livers of the two groups of rats demonstrated no differences between rats treated with DCLF alone and those treated with DCLF following antibiotic pretreatment, suggesting that the protective effects of antibiotics were not due to changes in the expression of large numbers of genes. However, these data did reveal a relatively small number of genes that were differentially expressed between the Published online 12/15/2008 • DOI: 10.1021/tx800412w © 2008 American Chemical Society
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to the mitochondria, which soon thereafter became rounded and deformed. Rigoni et al. next directly studied the effects of SPANs on isolated rat brain mitochondria. They found that SPAN exposure facilitated the opening of the mitochondrial permeability transition pore (PTP) and that this effect correlated with the phospholipase activity of each individual SPAN. Because PTP opening results in a failure of mitochondria to take up and store Ca2+ from the cytosol, this effect helps to explain the increase in cytosolic Ca2+ associated with SPAN intoxication. Furthermore, because SPAN activity is Ca2+-dependent, the opening of the PTP facilitates further SPANmediated phospholipid hydrolysis and the resulting cellular damage. Thus, it appears that the mitochondrion is an important key to SPAN neurotoxicity. •Carol A. Rouzer
two groups. Functional analysis of these genes showed an association between the antibiotic treatment and the regulation of the retinoid X, farnesoid X, and peroxisome proliferator-activated receptors, along with fatty acid metabolism and acute phase response proteins. Because some of these pathways are also altered by LPS treatment, these data support the hypothesis that the antibiotics protected against DCLF hepatotoxicity by preventing LPS or bacterial exposure. Consistent with a possible role for the inflammatory response, an accumulation of neutrophils was observed in the livers of DCLF-treated rats. However, pretreatment of the rats with an antineutrophil antibody to eliminate the neutrophil accumulation had no effect on hepatotoxicity.
Figure kindly provided by Robert Roth.
Expression analysis also revealed a group of genes that were altered by DCLF exposure regardless of antibiotic pretreatment. The finding that many of these genes were associated with the response to oxidative stress and hyVol. 21,
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Spotlight poxia led Deng et al. to demonstrate that a hepatotoxic dose of DCLF induces a hypoxic state in the liver. They further showed that hypoxia exacerbates DCLF-induced cytotoxicity in cultured hepatocytes. Together, the data support a role for bacteria in DCLFmediated hepatotoxicity, although it is also likely that DCLF alters homeostasis of hepatocytes to render them more sensitive to injury. Because the bacterial contribution may be expected to be sporadic and unpredictable, this phenomenon may help to explain the idiosyncratic nature of DCLF-induced hepatotoxicity in the clinic. •Carol A. Rouzer
Exploring Mutations by SAMS Damage to DNA bases may lead to mutations if the damage results in replication errors. Much work has been done to determine the effect of specific lesions on DNA replication, but this effort has been confounded by the fact that the outcome of DNA replication of a damaged base is influenced by its sequence context. Now, Fang and Taylor [(2008) Nucl. Acids Res. 36, 6004] address this problem with a new high-throughput method to evaluate the effect of sequence context on the replication of DNA across a damage site.
Reproduced with permission from Fang and Taylor [(2008) Nucl. Acids Res. 36, 6004]. Copyright 2008 Oxford University Press.
Called the serial analysis of mutation spectra (SAMS) approach, the method requires the synthesis of a random library of templates, all containing a chemically defined damaged base at a defined site near the center and differing only with regard to the fact that the bases on either side of the damage site are randomly assigned. A primer is designed so that replication of the template produces bypass product strands that are shorter than the template, allowing them to be separated by size. The replicated strand is then amplified by PCR and cut with a restriction enzyme. The fragments are polymerized through annealing and ligation of the complementary ends generated by the restriction endonuclease. Insertion of these concatenated product DNAs into a vector allows cloning, replication, and sequencing. Multiple bypass product sequences can then be extracted from each clone, increasing throughput. The method theoretically provides se2226
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quence data on the replication of every possible combination of bases flanking the damage site. This goal will not be realized if some sequence combinations block replication or result in deletion products that are eliminated during the isolation of the product strands. Fang and Taylor applied this method to the replication of templates bearing a synthetic tetrahydrofuran abasic site by the yeast polymerase pol η. The results provided a wealth of data concerning the effect of neighboring bases on the production of substitution and deletion mutations, allowing the authors to propose mechanisms by which these errors could occur. These promising findings indicate the power of the method, which may be expanded to increase the number of randomized bases. •Carol A. Rouzer
Redox Balance in Small Spaces Maintenance of intracellular redox balance is an important aspect of homeostasis, as well as a key to the cell’s ability to respond to oxidative stress. It has long been recognized that the oxidation potential varies among different cellular organelles, but accurate elucidation of these varying conditions has been challenging. Now, Hu et al. [(2008) J. Biol. Chem. 283, 29126] have found a new way to meet that challenge, by designing a green fluorescent proteinbased redox sensor (rxYFP) targeted exclusively to the cytosol, mitochondrial matrix, or mitochondrial intermembrane space (IMS). Selective expression of each of the targeted rxYFP proteins in yeast demonstrated a distinct redox environment for each compartment. The responsiveness of the rxYFP proteins to external stressors was confirmed by treating the cells with the oxidant 4,4′-dithiodipyridine or the reductant dithiothreitol. Expression of the targeted rxYFP sensors in cells lacking glutathione reductase (Glr1) revealed that the redox status of the mitochondrial matrix and cytosol, but not the IMS, was highly dependent on this enzyme. Targeted expression of Glr1 to the mitochondrial matrix or the cytosol in glr1∆ cells demonstrated selective regulation of the redox status of matrix-rxYFP and cytosolicrxYFP. These data demonstrate the value of the use of targeted rxYFP expression to monitor intraorganelle redox status in intact cells and confirm independent regulation of this important parameter, even within subcompartments of an organelle. The relative oxidizing environment of the IMS discovered here may be important in the transport of proteins into the IMS, which has been shown to be dependent on disulfide oxidation within the IMS compartment. Although the exact mechanisms for the maintenance of redox differences between organelles are not known, these new techniques provide an important tool for further exploration of these important questions. •Carol A. Rouzer TX800412W Published online 12/15/2008 •
DOI: 10.1021/tx800412w $40.75 © 2008 American Chemical Society
