Su(var)3-9 mediates age-dependent increase in H3K9 methylation on TDP-43 promoter triggering neurodegeneration.

Aging progressively modifies the physiological balance of the organism increasing susceptibility to both genetic and sporadic neurodegenerative diseases. These changes include epigenetic chromatin remodeling events that may modify the transcription levels of disease-causing genes affecting neuronal survival. However, how these events interconnect is not well understood. Here, we found that Su(var)3-9 causes increased methylation of histone H3K9 in the promoter region of TDP-43, the most frequently altered factor in amyotrophic lateral sclerosis (ALS), affecting the mRNA and protein expression levels of this gene through epigenetic modifications that appear to be conserved in aged Drosophila brains, mouse, and human cells. Remarkably, augmented Su(var)3-9 activity causes a decrease in TDP-43 expression followed by early defects in locomotor activities. In contrast, decreasing Su(var)3-9 action promotes higher levels of TDP-43 expression, improving motility parameters in old flies. The data uncover a novel role of this enzyme in regulating TDP-43 expression and locomotor senescence and indicate conserved epigenetic mechanisms that may play a role in the pathogenesis of ALS.

Moonlighting in Mitosis: Analysis of the Mitotic Functions of Transcription and Splicing Factors.

Moonlighting proteins can perform one or more additional functions besides their primary role. It has been posited that a protein can acquire a moonlighting function through a gradual evolutionary process, which is favored when the primary and secondary functions are exerted in different cellular compartments. Transcription factors (TFs) and splicing factors (SFs) control processes that occur in interphase nuclei and are strongly reduced during cell division, and are therefore in a favorable situation to evolve moonlighting mitotic functions. However, recently published moonlighting protein databases, which comprise almost 400 proteins, do not include TFs and SFs with secondary mitotic functions. We searched the literature and found several TFs and SFs with bona fide moonlighting mitotic functions, namely they localize to specific mitotic structure(s), interact with proteins enriched in the same structure(s), and are required for proper morphology and functioning of the structure(s). In addition, we describe TFs and SFs that localize to mitotic structures but cannot be classified as moonlighting proteins due to insufficient data on their biochemical interactions and mitotic roles. Nevertheless, we hypothesize that most TFs and SFs with specific mitotic localizations have either minor or redundant moonlighting functions, or are evolving towards the acquisition of these functions.

Neurotoxicity of antineoplastic drugs: Mechanisms, susceptibility, and neuroprotective strategies.

This review summarizes the adverse effects on the central and/or peripheral nervous systems that may occur in response to antineoplastic drugs. In particular, we describe the neurotoxic side effects of the most commonly used drugs, such as platinum compounds, doxorubicin, ifosfamide, 5-fluorouracil, vinca alkaloids, taxanes, methotrexate, bortezomib and thalidomide. Neurotoxicity may result from direct action of compounds on the nervous system or from metabolic alterations produced indirectly by these drugs, and either the central nervous system or the peripheral nervous system, or both, may be affected. The incidence and severity of neurotoxicity are principally related to the dose, to the duration of treatment, and to the dose intensity, though other factors, such as age, concurrent pathologies, and genetic predisposition may enhance the occurrence of side effects. To avoid or reduce the onset and severity of these neurotoxic effects, the use of neuroprotective compounds and/or strategies may be helpful, thereby enhancing the therapeutic effectiveness of antineoplastic drug.

RNAi-mediated depletion of the NSL complex subunits leads to abnormal chromosome segregation and defective centrosome duplication in Drosophila mitosis.

The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins.

The role of Patronin in Drosophila mitosis.

BACKGROUND: The calmodulin-regulated spectrin-associated proteins (CAMSAPs) belong to a conserved protein family, which includes members that bind the polymerizing mcrotubule (MT) minus ends and remain associated with the MT lattice formed by minus end polymerization. Only one of the three mammalian CAMSAPs, CAMSAP1, localizes to the mitotic spindle but its function is unclear. In Drosophila, there is only one CAMSAP, named Patronin. Previous work has shown that Patronin stabilizes the minus ends of non-mitotic MTs and is required for proper spindle elongation. However, the precise role of Patronin in mitotic spindle assembly is poorly understood. RESULTS: Here we have explored the role of Patronin in Drosophila mitosis using S2 tissue culture cells as a model system. We show that Patronin associates with different types of MT bundles within the Drosophila mitotic spindle, and that it is required for their stability. Imaging of living cells expressing Patronin-GFP showed that Patronin displays a dynamic behavior. In prometaphase cells, Patronin accumulates on short segments of MT bundles located near the chromosomes. These Patronin "seeds" extend towards the cell poles and stop growing just before reaching the poles. Our data also suggest that Patronin localization is largely independent of proteins acting at the MT minus ends such as Asp and Klp10A. CONCLUSION: Our results suggest a working hypothesis about the mitotic role of Patronin. We propose that Patronin binds the minus ends within MT bundles, including those generated from the walls of preexisting MTs via the augmin-mediated pathway. This would help maintaining MT association within the mitotic bundles, thereby stabilizing the spindle structure. Our data also raise the intriguing possibility that the minus ends of bundled MTs can undergo a limited polymerization.

Publisher Correction: The role of Patronin in Drosophila mitosis.

During production of the original article [1], there was a technical error that resulted in author corrections not being rendered in the PDF version of the article.

Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation.

Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells.

The Drosophila orthologue of the INT6 onco-protein regulates mitotic microtubule growth and kinetochore structure.

INT6/eIF3e is a highly conserved component of the translation initiation complex that interacts with both the 26S proteasome and the COP9 signalosome, two complexes implicated in ubiquitin-mediated protein degradation. The INT6 gene was originally identified as the insertion site of the mouse mammary tumor virus (MMTV), and later shown to be involved in human tumorigenesis. Here we show that depletion of the Drosophila orthologue of INT6 (Int6) results in short mitotic spindles and deformed centromeres and kinetochores with low intra-kinetochore distance. Poleward flux of microtubule subunits during metaphase is reduced, although fluorescence recovery after photobleaching (FRAP) demonstrates that microtubules remain dynamic both near the kinetochores and at spindle poles. Mitotic progression is delayed during metaphase due to the activity of the spindle assembly checkpoint (SAC). Interestingly, a deubiquitinated form of the kinesin Klp67A (a putative orthologue of human Kif18A) accumulates near the kinetochores in Int6-depleted cells. Consistent with this finding, Klp67A overexpression mimics the Int6 RNAi phenotype. Furthermore, simultaneous depletion of Int6 and Klp67A results in a phenotype identical to RNAi of just Klp67A, which indicates that Klp67A deficiency is epistatic over Int6 deficiency. We propose that Int6-mediated ubiquitination is required to control the activity of Klp67A. In the absence of this control, excess of Klp67A at the kinetochore suppresses microtubule plus-end polymerization, which in turn results in reduced microtubule flux, spindle shortening, and centromere/kinetochore deformation.

Mechanisms of Neuroprotection by Quercetin: Counteracting Oxidative Stress and More.

Increasing interest has recently focused on determining whether several natural compounds, collectively referred to as nutraceuticals, may exert neuroprotective actions in the developing, adult, and aging nervous system. Quercetin, a polyphenol widely present in nature, has received the most attention in this regard. Several studies in vitro, in experimental animals and in humans, have provided supportive evidence for neuroprotective effects of quercetin, either against neurotoxic chemicals or in various models of neuronal injury and neurodegenerative diseases. The exact mechanisms of such protective effects remain elusive, though many hypotheses have been formulated. In addition to a possible direct antioxidant effect, quercetin may also act by stimulating cellular defenses against oxidative stress. Two such pathways include the induction of Nrf2-ARE and induction of the antioxidant/anti-inflammatory enzyme paraoxonase 2 (PON2). In addition, quercetin has been shown to activate sirtuins (SIRT1), to induce autophagy, and to act as a phytoestrogen, all mechanisms by which quercetin may provide its neuroprotection.

Role of glutamate receptors in tetrabrominated diphenyl ether (BDE-47) neurotoxicity in mouse cerebellar granule neurons.

The polybrominated diphenyl ether (PBDE) flame retardants are developmental neurotoxicants, as evidenced by numerous in vitro, animal and human studies. PBDEs can alter the homeostasis of thyroid hormone and directly interact with brain cells. Induction of oxidative stress, leading to DNA damage and apoptotic cell death is a prominent mechanism of PBDE neurotoxicity, though other mechanisms have also been suggested. In the present study we investigated the potential role played by glutamate receptors in the in vitro neurotoxicity of the tetrabromodiphenyl ether BDE-47, one of the most abundant PBDE congeners. Toxicity of BDE-47 in mouse cerebellar neurons was diminished by antagonists of glutamate ionotropic receptors, but not by antagonists of glutamate metabotropic receptors. Antagonists of NMDA and AMPA/Kainate receptors also inhibited BDE-47-induced oxidative stress and increases in intracellular calcium. The calcium chelator BAPTA-AM also inhibited BDE-47 cytotoxicity and oxidative stress. BDE-47 caused a rapid increase of extracellular glutamate levels, which was not antagonized by any of the compounds tested. The results suggest that BDE-47, by still unknown mechanisms, increases extracellular glutamate which in turn activates ionotropic glutamate receptors leading to increased calcium levels, oxidative stress, and ultimately cell death.

Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex [corrected].

Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and ß-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.

The brominated flame retardant BDE-47 causes oxidative stress and apoptotic cell death in vitro and in vivo in mice.

Polybrominated diphenyl ethers (PBDEs), used for decades as flame retardants, have become widespread environmental contaminants. Exposure is believed to occur primarily through diet and dust, and infants and toddlers have the highest body burden, raising concern for potential developmental neurotoxicity. The exact mechanisms of PBDE neurotoxicity have not been elucidated, but two relevant modes of action relate to impairment of thyroid hormone homeostasis and to direct effects on brain cells causing alterations in signal transduction, oxidative stress and apoptotic cell death. The present study shows that BDE-47 (2,2',4,4'-tetrabromodiphenyl ether) induces oxidative stress and ensuing apoptotic cell death in mouse cerebellar granule neurons in vitro. Similarly, in vivo administration of BDE-47, according to an exposure protocol shown to induce behavioral and biochemical alterations (10mg/kg, per os on post-natal day 10), induces oxidative stress and apoptosis, without altering serum levels of thyroid hormones. The effects of BDE-47 both in vitro and in vivo were more pronounced in a mouse model lacking the modifier subunit of glutamate cysteine ligase (GCLM) which results in reduced anti-oxidant capability due to low levels of GSH. Concentrations of BDE-47 in brain were in the mid-nanomolar range. These findings indicate that effects observed with BDE-47 in vitro are also present after in vivo administration, suggesting that in addition to potential endocrine effects, which were not seen here, direct interactions with brain cells should be considered as a potential mechanism of BDE-47 neurotoxicity.

Comparison of drought stress response and gene expression between a GM maize variety and a near-isogenic non-GM variety.

Maize MON810, grown and commercialised worldwide, is the only cultivated GM event in the EU. Maize MON810, variety DKC6575, and the corresponding near-isogenic line Tietar were studied in different growth conditions, to compare their behaviour in response to drought. Main photosynthetic parameters were significantly affected by drought stress in both GM and non-GM varieties to a similar extent. Though DKC6575 (GM) had a greater sensitivity in the early phase of stress response as compared with Tietar (non-GM), after six days of stress they behaved similarly, and both varieties recovered from stress damage. Profiling gene expression in water deficit regimes and in a generalised drought stress condition showed an up-regulation of many stress-responsive genes, but a greater number of differentially expressed genes was observed in Tietar, with genes belonging to transcription factor families and genes encoding heat shock proteins, late embryogenesis abundant proteins and detoxification enzymes. Since induction of these genes have been indicated from the literature as typical of stress responses, their activation in Tietar rather than in DKC6575 may be reminiscent of a more efficient response to drought. DKC6575 was also analysed for the expression of the transgene CryIAb (encoding the delta-endotoxin insecticidal protein) in water deficit conditions. In all the experiments, the CryIAb transcript was not influenced by drought stress, but was expressed at a constant level. This suggests that though possessing a different pattern of sensitivity to stress, the GM variety maintains the same expression level for the transgene.

Characterization of a melamino nitroheterocycle as a potential lead for the treatment of human african trypanosomiasis.

This paper reports an evaluation of a melamino nitroheterocycle, a potential lead for further development as an agent against human African trypanosomiasis (HAT). Studies on its efficacy, physicochemical and biopharmaceutical properties, and potential for toxicity are described. The compound previously had been shown to possess exceptional activity against Trypanosoma brucei in in vitro assays comparable to that of melarsoprol. Here, we demonstrate that the compound also was curative in the stringent acute mouse model T. brucei rhodesiense STIB 900 when given intraperitoneally at 40 mg/kg of body weight. Nevertheless, activity was only moderate when the oral route was used, and no cure was obtained when the compound was tested in a stage 2 rodent model of infection. Genotoxic profiling revealed that the compound induces DNA damage by a mechanism apparently independent from nitroreduction and involving the introduction of base pair substitutions (Ames test), possibly caused by oxidative damage of the DNA (comet test). No significant genotoxicity was observed at the chromosome level (micronucleus assay). The lack of suitable properties for oral and central nervous system uptake and the genotoxic liabilities prevent the progression of this melamine nitroheterocycle as a drug candidate for HAT. Further modification of the compound is required to improve the pharmacokinetic properties of the molecule and to separate the trypanocidal activity from the toxic potential.

Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation.

The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. We have tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. Our work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules.

A mechanistic view of polybrominated diphenyl ether (PBDE) developmental neurotoxicity.

Polybrominated diphenyl ethers (PBDEs), extensively used in the past few decades as flame retardants in a variety of consumer products, have become world-wide persistent environmental pollutants. Levels in North America are usually higher than those in Europe and Asia, and body burden is 3-to-9-fold higher in infants and toddlers than in adults. The latter has raised concern for potential developmental toxicity and neurotoxicity of PBDEs. Experimental studies in animals and epidemiological observations in humans suggest that PBDEs may be developmental neurotoxicants. Pre- and/or post-natal exposure to PBDEs may cause long-lasting behavioral abnormalities, particularly in the domains of motor activity and cognition. The mechanisms underlying the developmental neurotoxic effects of PBDEs are not known, though several hypotheses have been put forward. One general mode of action relates to the ability of PBDEs to impair thyroid hormone homeostasis, thus indirectly affecting the developing brain. An alternative or additional mode of action involves a direct effect of PBDEs on nervous system cells; PBDEs can cause oxidative stress-related damage (DNA damage, mitochondrial dysfunction, apoptosis), and interfere with signal transduction (particularly calcium signaling), and with neurotransmitter systems. Important issues such as bioavailability and metabolism of PBDEs, extrapolation of results to low level of exposures, and the potential effects of interactions among PBDE congeners and between PBDEs and other contaminants also need to be taken into account.

Paraoxonase-2 (PON2) in brain and its potential role in neuroprotection.

Paraoxonase 2 (PON2) is a member of a gene family which also includes the more studied PON1, as well as PON3. PON2 is unique among the three PONs, as it is expressed in brain tissue. PON2 is a lactonase and displays anti-oxidant and anti-inflammatory properties. PON2 levels are highest in dopaminergic regions (e.g. striatum), are higher in astrocytes than in neurons, and are higher in brain and peripheral tissues of female mice than male mice. At the sub-cellular level, PON2 localizes primarily in mitochondria, where it scavenges superoxides. Lack of PON2 (as in PON2(-/-) mice), or lower levels of PON2 (as in male mice compared to females) increases susceptibility to oxidative stress-induced toxicity. Estradiol increases PON2 expression in vitro and in vivo, and provides neuroprotection against oxidative stress. Such neuroprotection is not present in CNS cells from PON2(-/-) mice. Similar results are also found with the polyphenol quercetin. PON2, given its cellular localization and antioxidant and anti-inflammatory actions, may represent a relevant enzyme involved in neuroprotection, and may represent a novel target for neuroprotective strategies. Its differential expression in males and females may explain gender differences in the incidence of various diseases, including neurodevelopmental, neurological, and neurodegenerative diseases.

Modulation of paraoxonase 2 (PON2) in mouse brain by the polyphenol quercetin: a mechanism of neuroprotection?

Quercetin is a common flavonoid polyphenol which has been shown to exert neuroprotective actions in vitro and in vivo. Though quercetin has antioxidant properties, it has been suggested that neuroprotection may be ascribed to its ability of inducing the cell's own defense mechanisms. The present study investigated whether quercetin could increase the levels of paraoxonase 2 (PON2), a mitochondrial enzyme expressed in brain cells, which has been shown to have potent antioxidant properties. PON2 protein, mRNA, and lactonase activity were highest in mouse striatal astrocytes. Quercetin increased PON2 levels, possibly by activating the JNK/AP-1 pathway. The increased PON2 levels induced by quercetin resulted in decreased oxidative stress and ensuing toxicity induced by two oxidants. The neuroprotective effect of quercetin was significantly diminished in cells from PON2 knockout mice. These findings suggest that induction of PON2 by quercetin represents an important mechanism by which this polyphenol may exert its neuroprotective action.

Bypassing the Greatwall-Endosulfine pathway: plasticity of a pivotal cell-cycle regulatory module in Drosophila melanogaster and Caenorhabditis elegans.

In vertebrates, mitotic and meiotic M phase is facilitated by the kinase Greatwall (Gwl), which phosphorylates a conserved sequence in the effector Endosulfine (Endos). Phosphorylated Endos inactivates the phosphatase PP2A/B55 to stabilize M-phase-specific phosphorylations added to many proteins by cyclin-dependent kinases (CDKs). We show here that this module functions essentially identically in Drosophila melanogaster and is necessary for proper mitotic and meiotic cell division in a wide variety of tissues. Despite the importance and evolutionary conservation of this pathway between insects and vertebrates, it can be bypassed in at least two situations. First, heterozygosity for loss-of-function mutations of twins, which encodes the Drosophila B55 protein, suppresses the effects of endos or gwl mutations. Several types of cell division occur normally in twins heterozygotes in the complete absence of Endos or the near absence of Gwl. Second, this module is nonessential in the nematode Caenorhaditis elegans. The worm genome does not contain an obvious ortholog of gwl, although it encodes a single Endos protein with a surprisingly well-conserved Gwl target site. Deletion of this site from worm Endos has no obvious effects on cell divisions involved in viability or reproduction under normal laboratory conditions. In contrast to these situations, removal of one copy of twins does not completely bypass the requirement for endos or gwl for Drosophila female fertility, although reducing twins dosage reverses the meiotic maturation defects of hypomorphic gwl mutants. These results have interesting implications for the function and evolution of the mechanisms modulating removal of CDK-directed phosphorylations.

Evaluation of DNA damage induced by 2 polybrominated diphenyl ether flame retardants (BDE-47 and BDE-209) in SK-N-MC cells.

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants whose levels have increased in the environment and in human tissues in the past decades. Exposure to PBDEs has been associated with developmental neurotoxicity, endocrine dysfunction, and reproductive disorders. In spite of their widespread distribution and potential adverse health effects, only few studies have addressed the potential neurotoxicity of PBDEs. In the present study, we evaluated the cyto- and genotoxicity of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and decabrominated diphenyl ether (BDE-209) in human neuroblastoma cells (SK-N-MC). The DNA damage was measured using the alkaline version of the Comet assay, while specific oxidative-generated DNA damage was evaluated by a modified version of the Comet assay with the repair enzyme formamidopyrimidine glycosylase (FPG). The results show that BDE-47 and BDE-209 (5-20 µmol/L) are able to induce DNA damage in human SK-N-MC cells. Pretreatment with the antioxidant melatonin significantly reduced the DNA damage induced by both congeners. The Comet assay carried out in the presence of FPG suggests that both congeners increase purine oxidation. In all cases, BDE-47 was more potent than BDE-209. The results indicate that 2 environmentally relevant PBDEs cause DNA damage which is primarily mediated by the induction of oxidative stress and may contribute to adverse health effects.