In This Issue pubs.acs.org/crt
■
SPECIAL FEATURES Metabolism of a drug by a single enzyme is a generally accepted risk factor for drug−drug interactions. Now, Isoherranen et al. (DOI: 10.1021/tx300192g) challenge this concept by identifying multi-P450 inhibitors and demonstrating that these drugs can exhibit significant drug−drug interactions with well-known drugs that are also cleared by multiple pathways. Do not miss this chance to challenge your assumptions! Mesoporous silica nanoparticles, notable for their tunable sizes, wide range of shapes, high surface areas, and readily functionalizable surfaces, offer numerous potential pharmaceutical and biological applications. Thus, the biocompatibility of these particles is of great interest. To learn more about this important topic, check out the timely review by Asefa and Tao (DOI: 10.1021/tx300166u).
■
NEW DETOXICATING ACTIVITY FOR MARC
because both showed inhibition of the N-reductive activity with concentrations above 1 mM N6-hydroxyadenine. Together, the data suggest that the mARCs are likely the most important enzymes for the detoxication of N-hydroxylated nucleobases and nucleosides. Although these enzymes may perform other functions, this is likely one of their most physiologically significant roles within the cell.
■
Reaction of nucleobases and/or their corresponding nucleosides or nucleotides with peroxyradicals can lead to Nhydroxylated products. If incorporated into DNA and not repaired, these can be highly mutagenic lesions. Thus, their detoxication pathway is of great interest, leading Krompholz et al. (DOI: 10.1021/tx300298m) to explore the ability of the recently discovered mitochondrial amidoxime reducing component mARC enzymes (mARC1 and mARC2) to catalyze the reduction of N6-hydroxyadenine and N4-hydroxycytosine and their corresponding nucleosides. The mARCs, along with three other enzymes, are the only known mammalian proteins to contain molybdenum coordinated with 5,6,7,8-tetrahydropyranopterin as a cofactor. The mARCs work together with NADH cytochrome b5 reductase (cyt b5R) and cytochrome b5 (cyt b5) to transfer electrons from NADH to substrate N-hydroxylated compounds. Their role in the metabolism of amidoxime prodrugs is well established, but the possibility that they may serve in the detoxication of endogenously generated hydroxylated nucleobases has not been explored. Krompholz et al. prepared subcellular fractions from porcine liver. They found that reduction of N-hydroxylated nucleobases and nucleosides by these fractions was NADH-dependent and that the activity was the highest in the mitochondrial fraction. Comparison of mitochondria from multiple porcine tissues revealed the highest activity in thyroid, kidney, pancreas, and liver. Indeed, enrichment of the reduction of the Nhydroxylated nucleobases and nucleosides corresponded to the enrichment of the reduction of the mARC marker substrate benzamidoxime and the presence of mARC1 and mARC2 as detected by immunoblot analysis. Recombinant mARC1 and mARC2 reconstituted with cyt b5R, cyt b5, and NADH also catalyzed the reduction of the Nhydroxylated nucleobases and nucleosides. In general, mARC1 showed higher catalytic efficiency than mARC2. Although both enzymes were present, the catalytic behavior of porcine liver mitochondrial fractions more closely resembled that of mARC1 © 2012 American Chemical Society
ARSENIC AND THE TELOMERE
Telomeres are tandem repeat sequences found at the end of eukaryotic chromosomes. They are shortened with each cell division, and sufficiently short telomers signal the cell to stop dividing. Thus, telomeres serve as a mechanism to limit the life span of a cell. One way that cancer cells evade mortality is to lengthen their telomeres through the action of telomere reverse transcriptase encoded by the TERT gene. Arsenic is a strong carcinogen but weak mutagen, leading Li et al. (DOI: 10.1021/tx300222t) to hypothesize that a mechanism of arsenic carcinogenicity is to increase telomere length. They tested this hypothesis in a population of 202 women living in Argentina who were exposed to As in their drinking water at widely varying concentrations (from 3.5 to 200 μg/L). Inorganic arsenic (iAs) is metabolized via a series of methylations and reductions yielding methylarsonic acid (MMA) and dimethylarsinic acid (DMA), which, along with iAs, are excreted in the urine. A higher urinary DMA/MMA ratio is indicative of more rapid metabolism and detoxication. Li et al. measured iAs, DMA, and MMA in the urine and telomere length in the peripheral blood cells of their cohort of 202 women. Published: November 19, 2012 2261
dx.doi.org/10.1021/tx300414q | Chem. Res. Toxicol. 2012, 25, 2261−2262
Chemical Research in Toxicology
In This Issue
disease (PD), a neurodegenerative condition affecting the nitgrostrial system in the brain. Viquez et al. treated rats for 4 weeks with 0.3 mmol/kg N,Ndiethyldithiocarbamate (DEDC), a compound known to be metabolized to reactive electrophiles. Data-dependent LC-MS/ MS analysis of E1 isolated from brain protein extracts of treated and control rats readily identified the protein and revealed monoethyl adducts at Cys-234 and Cys-179. E1 activity results in the transfer of a ubiquitin molecule to the enzyme’s active site, producing activated E1. Although neither C-234 nor C179 is the residue to which ubiquitin is attached in activated E1, these residues are located in a groove required for binding and positioning of ubiquitin prior to its adenylation and thioester linkage at the active site cysteine. Thus, adduction of these residues may interfere with critical ubiquitin−E1 interactions. Consistently, the slot blot assay indicated that DEDC treatment resulted in reduced levels of ubiquitinated proteins and decreased levels of activated E1 in rat brain homogenates. In vitro incubation of recombinant E1 with ethylisocyanate, an ultimate electrophilic metabolite of DEDC, led to enzyme inactivation. The results suggested that DEDC-mediated E1 adduction results in the inhibition of E1 activity and protein ubiquitination in the brain, leading Viquez et al. to evaluate the broader effects of DEDC. Global protein expression analysis identified increased and decreased expression of 9 proteins and 1 protein, respectively, in DEDC-treated rat brains. These included myelin structural proteins, antioxidant proteins, and one protein involved in catechol synthesis, which is also frequently increased in PD. Protein carbonyl content increased in homogenates from the striatum, but not whole brain, suggesting elevated oxidative damage in that brain region. Elevations in the expression of the antioxidant enzymes heme oxygenase-1 and superoxide dismutase-1 in the striatum further supported that conclusion. An important neurotransmitter released by axons projecting from the substantia nigra to the striatum is dopamine (DA). Consistent with DEDC-mediated damage to this region of the brain, animals treated with the agent showed reduced levels of the DA synthetic enzyme tyrosine hydroxylase (TH). However, levels of activated TH, as indicated by protein phosphorylation, were increased in DEDC-treated animals, and DA levels were normal, suggesting a compensatory response to the neurotoxic damage. Increased levels of the DA transporter were also consistent with a perturbation of DA homeostasis. A hallmark of PD is the accumulation of protein aggregates containing α-synuclein and phosphorylated tau. Increased levels of both of these proteins in the striatum of DEDC-treated rats would facilitate oligomer formation in the long term. The results suggest that E1 is susceptible to electrophilic adduction and that the S-monoethylation of Cys-234 and/or Cys-179 of E1 in DEDC-treated rats leads to inhibition of activity, ultimately setting the stage for neurotoxic changes similar to those seen in PD. Whether electrophilic damage of E1 contributes to sporadic cases of PD remains the subject of future research.
The results indicated a wide range of total urinary As (0.1 to 1251 μg/L, median 230 μg/L) and telomere length (0.18 to 0.67, median 0.37). Using Spearman’s rho correlation, Li et al. showed that telomere length was positively correlated with urinary iAs and inversely correlated with DMA. Linear regression analysis revealed a significant positive association between urinary As and telomere length. When the data were stratified by the median of the metabolites, an association was found only in the groups with a high fraction of iAs and/or MMA. These results suggest that high levels of urinary As, particularly when present in its more toxic forms, are correlated with increased telomere length. The enzyme primarily responsible for As methylation is arsenic (+3 oxidation state) methyltransferase (AS3MT). Li et al. genotyped a subset of their study population for 8 haplotypes of AS3MT. They found that subjects carrying a haplotype associated with slow metabolism and increased toxicity exhibited a stronger association between urinary As levels and telomere length. They also discovered that expression of TERT was positively associated with urinary As levels, though no association with telomere length was evident. Telomere length was positively associated with the expression of three genes and negatively associated with eight genes involved in telomere regulation, but only one of these genes RAP1B demonstrated a weak positive association with urinary As. The results support the hypothesis that one mechanism of As carcinogenicity is through the modulation of telomere length, enabling cells to evade the normal process of senescence and death. The correlation between As and telomere length is most prominent in individuals who demonstrate slow metabolic detoxication of As. Increased expression of TERT may be one mechanism by which As exerts its effect on telomere length, but further work will be needed to fully elucidate how modulation of telomere length contributes to As-mediated carcinogenicity.
■
ELECTROPHILE-MEDIATED NEUROTOXICITY
A recent study in HEK293 cells identified 90 cytosolic proteins that are highly susceptible to damage mediated by reactive electrophiles. One of these, ubiquitin activating enzyme E1 (E1), catalyzes the first step in the transfer of ubiquitin to target proteins, which initiates ubiquitin-dependent protein processing. A wide array of cellular functions, including protein trafficking and degradation, modulation of transcription factor activity, and regulation of cell signaling, rely on ubiquitin-based processes, so Viquez et al. (DOI: 10.1021/tx300198h) investigated the effects of electrophile-mediated modification of E1 in vivo. They focused their attention on the central nervous system because mutations in ubiquitin pathway enzymes are associated with familial forms of Parkinson’s 2262
dx.doi.org/10.1021/tx300414q | Chem. Res. Toxicol. 2012, 25, 2261−2262
