Spotlight pubs.acs.org/crt
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ALKB DIOXYGENASE PREFERENTIALLY REPAIRS PROTONATED SUBSTRATES DNA adducts formed from the reaction of nucleobases with exogenous or endogenous electrophiles often cause miscoding or blockage of DNA replication, resulting in mutation or cell death. However, adducted DNA may be repaired using a variety of pathways, such as excision of the damaged nucleobase from the DNA or direct removal of an alkylation from the damaged base. AlkB dioxygenase performs the latter by oxidizing alkyl groups or exocyclic DNA rings, which are then spontaneously released from the nucleobase. Due to known differences in repair efficiencies among AlkB substrates, it was hypothesized that AlkB preferably recognizes and repairs protonated substrates. Agnieszka Maciejewska and co-workers recently reported a combination of experimental and computational studies that tested this hypothesis ((2013) J. Biol. Chem., 288, 432−431). They measured the rates of E. coli AlkB-mediated repair of several DNA adducts at various pH values and concentrations of Fe(II) ions and α-ketoglutarate (αKG), two cofactors required for AlkB activity. At a pH of 7.5, substrates with pKa values above 7.5 were at least 90% protonated and were repaired one-to-2 orders of magnitude faster than substrates with pKa values below 7.5. Under acidic conditions, the reaction rates for the latter substrates improved slightly due to better binding of the substrates to the AlkB active site, an observation which the authors confirmed by molecular modeling. However, reactions in acidic conditions required high concentrations of Fe(II) to reach optimum efficiency due to decreased Fe(II) binding in the enzyme active center at low pH. Meanwhile, optimal αKG concentration did not correlate with pH since even at the most acidic conditions screened, the cofactor maintained its active doubly dissociated anionic form. Collectively, the authors found that the best AlkB substrates are those which are mostly protonated at physiological pH, conditions allowing for ideal substrate and Fe(II) binding and αKG activity. Heidi A. Dahlmann
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FROM PESTICIDES TO PARKINSON’S DISEASE
zebrafish embryos, and that the compound (as well as its metabolites BIC and MBT acting via its sulfoxide MBT-SO) inhibited ALDH activity in neuronal cell cultures and isolated liver mitochondria. Dopaminergic neurons treated with benomyl showed substantially decreased levels of DOPAC, the product of ALDH-mediated oxidation of DOPAL. Furthermore, when DOPAL formation was suppressed in cells, the cytotoxicity of benomyl also decreased. On the basis of this evidence, the authors propose that benomyl metabolites inhibit ALDH activity, which prevents the conversion of DOPAL to DOPAC, and that the subsequent DOPAL accumulation causes the loss of dopaminergic neurons that occurs in PD. Heidi A. Dahlmann
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Millions of people worldwide suffer from Parkinson’s disease (PD), the symptoms of which stem from the progressive degeneration of dopaminergic neurons in the brain. Epidemiological data collected over decades indicate a link between PD occurrence and exposure to pesticides like the fungicide benomyl. Benomyl is known to both disrupt microtubule assembly and inhibit aldehyde dehydrogenase (ALDH) activity, biological consequences which have each been implicated in PD etiology. The latter effect offers a possible mechanism for benomyl-mediated PD pathogenesis since ALDH metabolizes the neurotoxic dopamine metabolite DOPAL. Inhibition of ALDH activity would result in toxic accumulation of DOPAL in dopaminergic neurons, which are then lost as PD progresses. Recent data collected by a multidisciplinary research team led by Arthur Fitzmaurice and Jeff Bronstein and including John Casida support this hypothesis ((2013) Proc. Natl. Acad. Sci., 110, 636−641). The team demonstrated that benomyl was toxic specifically to dopaminergic neurons both in vitro and in © 2013 American Chemical Society
LONG-TERM ALCOHOL EXPOSURE IMPAIRS ONE-CARBON METABOLISM AND DNA REPAIR IN BRAINS OF ADULT MICE
Two well-known consequences of chronic alcohol abuse are cognitive impairment and loss of neurons in the brain. It has been established that alcohol consumption leads to the generation of reactive oxygen species and acetaldehyde, which attack and form adducts with DNA. At the same time, longterm exposure to alcohol results in the inhibition of DNA repair due to impairment of one-carbon metabolism (OCM), which normally furnishes cells with DNA precursors and endogenous Published: February 28, 2013 303
dx.doi.org/10.1021/tx400075k | Chem. Res. Toxicol. 2013, 26, 303−304
Chemical Research in Toxicology
Spotlight
membrane potential (MMP) of cells treated with SWCNTs decreased, while the level of reactive oxygen species increased, indicating mitochondrial malfunction. Further experiments revealed that isolated Cyt c bound to and was subsequently reduced by SWCNTs in a pH-dependent manner. Using circular dichroism spectroscopy, the research team confirmed that the protein structure was altered in alkaline conditions, such that the active site became more exposed and able to be reduced by the SWCNTs. On the basis of these observations, the authors caution that the ability of SWCNTs to disrupt mitochondrial function needs to be carefully considered before SWCNTs can be used for in vivo applications. Heidi A. Dahlmann
methylating agents necessary for maintaining genomic stability. The link between these cellular-level disruptions and subsequent physiological changes in the brain has recently been further elaborated by Inna Kruman and co-workers ((2012) J. Biol. Chem., 287, 43533−43542). The research team began by confirming that prolonged exposure to alcohol resulted in increased levels of apoptosis markers and DNA damage products as well as activation of the DNA damage response in mice that were fed ethanolsupplemented liquid diets for three weeks, as compared with control mice that did not receive any alcohol. Mice treated with ethanol for three weeks also showed increased levels of homocysteine(Hcy), an established marker of OCM impairment, and decreased levels of global DNA methylation in the brain, while mice treated with ethanol for only four days showed no such signs of OCM disruption. The effect of longterm ethanol exposure was more pronounced in mice that were deficient in methylenetetrahydrofolate reductase (MTHFR), a key OCM enzyme. These mice displayed even greater levels of Hcy and compared to wild type mice treated with ethanol had lower capacity to repair DNA damage in the brain. The authors note that MTHFR mutations are common in humans and that this may lead to increased susceptibility to alcohol-induced neurodegeneration. Heidi A. Dahlmann
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SUPPRESSION OF OXIDATIVE STRESS BY β-HYDROXYBUTYRATE, AN ENDOGENOUS HISTONE DEACETYLASE INHIBITOR Gene expression is influenced in part through acetylation or deacetylation of histones, the core proteins around which genomic DNA is wrapped when condensed into chromosomes. The activities of certain classes of enzymes that perform histone deacetylation (HDACs) may be mediated by the concentrations of their endogenous cofactors, such as NAD+. However, endogenous regulators of zinc-dependent class I HDACs were unknown, until a recent report by Eric Verdin and co-workers ((2013) Science, 339, 211−214). β-Hydroxybutyrate (βOHB), a molecule that may reach millimolar concentrations in blood during prolonged fasting or diabetic ketoacidosis, is structurally similar to butyrate, a previously known exogenous class I HDAC inhibitor. Thus, Verdin and co-workers investigated the ability of βOHB to act as an HDAC inhibitor as well. Human embryonic kidney cells demonstrated dose-dependent increases in histone acetylation following βOHB treatment, while isolated recombinant human class I HDACs showed reduced deacetylase activity in vitro. In mice with elevated levels of βOHB (induced either by treatment with the molecule, by a prolonged fast, or by calorie restriction), certain histones were found to have significantly higher levels of acetylation. Consequently, specific genes, including those encoding oxidative stress resistance factors FOXO3A and MT2, were upregulated. The authors observed that βOHB-treated mice had enhanced resistance against oxidative stress and noted that this effect may be one reason for the health benefits conferred by calorie-restriction diets. Heidi A. Dahlmann
SINGLE-WALLED CARBON NANOTUBES ALTER CYTOCHROME C ELECTRON TRANSFER AND MODULATE MITOCHONDRIAL FUNCTION
Single-walled carbon nanotubes (SWCNTs), as biocompatible molecules with large surfaces that can be derivatized, have been investigated over the past decade as vehicles for drug, peptide, protein, plasmid, and siRNA delivery. Due to charge complementarity, pi-stacking, covalent bonding, and other nonspecific interactions, SWCNTs tend to bind strongly to proteins and other molecules. In many cases, this binding may alter protein structure and function. SWCNTs are also known to localize in mitochondria, but their impact on mitochondrial function, particularly by disruption of the mitochondrial membrane-bound protein cytochrome c (Cyt c), has previously been difficult to elucidate. Interactions between SWCNTs and Cyt c have been characterized by a team of researchers led by Ji-Hong Sun and Xing-Jie Liang ((2012) ACS Nano, 6, 10486−10496). The team verified that SWCNTs (modified with carboxy groups to improve solubility) permeated cells and accumulated in mitochondria, which consequently displayed greatly distorted morphology. The oxygen consumption and mitochondrial 304
dx.doi.org/10.1021/tx400075k | Chem. Res. Toxicol. 2013, 26, 303−304
