RESUMEN
Satellite DNA sequences are an integral part of centromeres, regions critical for faithful segregation of chromosomes during cell division. Because of their complex repetitive structure, satellite DNA may act as a barrier to DNA replication and other DNA based transactions ultimately resulting in chromosome breakage. Over the past two decades, several DNA repair proteins have been shown to bind and function at centromeres. While the importance of these repair factors is highlighted by various structural and numerical chromosome aberrations resulting from their inactivation, their roles in helping to maintain genome stability by solving the intrinsic difficulties of satellite DNA replication or promoting their repair are just starting to emerge. In this review, we summarize the current knowledge on the role of DNA repair and DNA damage response proteins in maintaining the structure and function of centromeres in different contexts. We also report the recent connection between the roles of specific DNA repair factors at these genomic loci with age-related increase of chromosomal instability under physiological and pathological conditions.
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Centrómero , ADN Satélite , Humanos , Centrómero/genética , ADN , Aneuploidia , Inestabilidad Genómica/genéticaRESUMEN
DNA/RNA synthesis precursors are especially vulnerable to damage induced by reactive oxygen species occurring following oxidative stress. Guanosine triphosphates are the prevalent oxidized nucleotides, which can be misincorporated during replication, leading to mutations and cell death. Here, we present a novel method based on micro-Raman spectroscopy, combined with ab initio calculations, for the identification, detection, and quantification of oxidized nucleotides at low concentration. We also show that the Raman signature in the terahertz spectral range (<100 cm-1) contains information on the intermolecular assembly of guanine in tetrads, which allows us to further boost the oxidative damage detection limit. Eventually, we provide evidence that similar analyses can be carried out on samples in very small volumes at very low concentrations by exploiting the high sensitivity of surface-enhanced Raman scattering combined with properly designed superhydrophobic substrates. These results pave the way for employing such advanced spectroscopic methods for quantitatively sensing the oxidative damage of nucleotides in the cell.
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Ácidos Nucleicos , Espectrometría Raman , Guanosina , Nucleótidos , Estrés OxidativoRESUMEN
Furan is a volatile compound that is formed in foods during thermal processing. It is classified as a possible human carcinogen by international authorities based on sufficient evidence of carcinogenicity from studies in experimental animals. Although a vast number of studies both in vitro and in vivo have been performed to investigate furan genotoxicity, the results are inconsistent, and its carcinogenic mode of action remains to be clarified. Here, we address the mutagenic and clastogenic activity of furan and its prime reactive metabolite cis-2 butene-1,4-dial (BDA) in mammalian cells in culture and in mouse animal models in a search for DNA lesions responsible of these effects. To this aim, Fanconi anemia-derived human cell lines defective in the repair of DNA inter-strand crosslinks (ICLs) and Ogg1-/- mice defective in the removal of 8-hydroxyguanine from DNA, were used. We show that both furan and BDA present a weak (if any) mutagenic activity but are clear inducers of clastogenic damage. ICLs are strongly indicated as key lesions for chromosomal damage whereas oxidized base lesions are unlikely to play a critical role.
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Aberraciones Cromosómicas/inducido químicamente , Furanos/efectos adversos , Mutación/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Carcinógenos , Línea Celular , Daño del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Furanos/toxicidad , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Micronúcleos con Defecto Cromosómico/inducido químicamente , Mutágenos , Oxidación-ReducciónRESUMEN
DNA trinucleotide repeat (TNR) expansion underlies several neurodegenerative disorders including Huntington's disease (HD). Accumulation of oxidized DNA bases and their inefficient processing by base excision repair (BER) are among the factors suggested to contribute to TNR expansion. In this study, we have examined whether oxidation of the purine dNTPs in the dNTP pool provides a source of DNA damage that promotes TNR expansion. We demonstrate that during BER of 8-oxoguanine (8-oxodG) in TNR sequences, DNA polymerase ß (POL ß) can incorporate 8-oxodGMP with the formation of 8-oxodG:C and 8-oxodG:A mispairs. Their processing by the OGG1 and MUTYH DNA glycosylases generates closely spaced incisions on opposite DNA strands that are permissive for TNR expansion. Evidence in HD model R6/2 mice indicates that these DNA glycosylases are present in brain areas affected by neurodegeneration. Consistent with prevailing oxidative stress, the same brain areas contained increased DNA 8-oxodG levels and expression of the p53-inducible ribonucleotide reductase. Our in vitro and in vivo data support a model where an oxidized dNTPs pool together with aberrant BER processing contribute to TNR expansion in non-replicating cells.
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ADN Glicosilasas/metabolismo , ADN/genética , ADN/metabolismo , Inestabilidad Genómica , Repeticiones de Trinucleótidos , 8-Hidroxi-2'-Desoxicoguanosina , Animales , ADN Polimerasa beta/metabolismo , Reparación del ADN , Desoxiguanosina/análogos & derivados , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Modelos Biológicos , Oxidación-Reducción , Estrés Oxidativo , Expansión de Repetición de TrinucleótidoRESUMEN
To determine prevalence, spectrum and genotype-phenotype correlations of MUTYH variants in Italian patients with suspected MAP (MUTYH-associated polyposis), a retrospective analysis was conducted to identify patients who had undergone MUTYH genetic testing from September 2002 to February 2014. Results of genetic testing and patient clinical characteristics were collected (gender, number of polyps, age at polyp diagnosis, presence of colorectal cancer (CRC) and/or other cancers, family data). The presence of large rearrangements of the MUTYH gene was evaluated by Multiplex Ligation-dependent Probe Amplification analysis. In all, 299 patients with colorectal neoplasia were evaluated: 61.2% were males, the median age at polyps or cancer diagnosis was 50 years (16-80 years), 65.2% had <100 polyps and 51.8% had CRC. A total of 36 different MUTYH variants were identified: 13 (36.1%) were classified as pathogenetic, whereas 23 (63.9%) were variants of unknown significance (VUS). Two pathogenetic variants were observed in 78 patients (26.1%). A large homozygous deletion of exon 15 was found in one patient (<1.0%). MAP patients were younger than those with negative MUTYH testing at polyps diagnosis (P<0.0001) and at first cancer diagnosis (P=0.007). MAP patients carrying the p.Glu480del variant presented with a younger age at polyp diagnosis as compared to patients carrying p.Gly396Asp and p.Tyr179Cys variants. A high heterogeneity of MUTYH variants and a high rate of VUS were identified in a cohort of Italian patients with suspected MAP. Genotype-phenotype analysis suggests that the p.Glu480del variant is associated with a severe phenotype.
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Pólipos del Colon/genética , ADN Glicosilasas/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Pólipos del Colon/patología , Neoplasias Colorrectales/genética , Femenino , Pruebas Genéticas , Genotipo , Humanos , Italia , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Mutación , Fenotipo , Estudios Retrospectivos , Adulto JovenRESUMEN
The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase ß (POL ß) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL ß incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL ß was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences.
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Disparidad de Par Base , ADN/química , Ribonucleótidos/química , ADN/metabolismo , ADN Polimerasa beta/metabolismo , Oxidación-Reducción , Ribonucleótidos/metabolismoRESUMEN
The MUTYH DNA glycosylase counteracts mutagenesis by removing adenine misincorporated opposite DNA 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated polyposis (MAP). The impact on genetic instability of the p.Tyr179Cys and p.Arg245His MUTYH variants was evaluated in lymphoblastoid cell lines (LCLs) derived from MAP patients and their relatives in comparison to wild-type LCLs. No difference in MUTYH expression was identified between wild type and LCLs with the p.Tyr179Cys, while the p.Arg245His mutation was associated with an unstable MUTYH protein. LCLs homozygous for the p.Tyr179Cys or the p.Arg245His variant contained increased DNA 8-oxodG levels and exhibited a mutator phenotype at the PIG-A gene. The extent of the increased spontaneous mutation frequency was 3-fold (range 1.6- to 4.6-fold) in four independent LCLs carrying the p.Tyr179Cys variant, while a larger increase (6-fold) was observed in two p.Arg245His LCLs. A similar hypermutability and S-phase delay following treatment with KBrO3 was observed in LCLs homozygous for either variant. When genetic instability was investigated in monoallelic p.Arg245His carriers, mutant frequencies showed an increase which is intermediate between wild-type and homozygous cells, whereas the mutator effect in heterozygous p.Tyr179Cys LCLs was similar to that in homozygotes. These findings indicate that the type of MUTYH mutation can affect the extent of genome instability associated with MUTYH inactivation. In addition, the mild spontaneous mutator phenotype observed in monoallelic carriers highlights the biological importance of this gene in the protection of the genome against endogenous DNA damage.
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Poliposis Adenomatosa del Colon/genética , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Inestabilidad Genómica , 8-Hidroxi-2'-Desoxicoguanosina , Poliposis Adenomatosa del Colon/sangre , Adulto , Bromatos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , Variación Genética , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Estabilidad Proteica , Adulto JovenRESUMEN
In checkpoint-deficient cells, DNA double-strand breaks (DSBs) are produced during replication by the structure-specific endonuclease MUS81. The mechanism underlying MUS81-dependent cleavage, and the effect on chromosome integrity and viability of checkpoint deficient cells is only partly understood, especially in human cells. Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate. Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81. Moreover, in CHK1-deficient cells depletion of RAD52, but not of MUS81, rescues chromosome instability observed after replication fork stalling. However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion. Our findings reveal a novel RAD52/MUS81-dependent mechanism that promotes cell viability and genome integrity in checkpoint-deficient cells, and disclose the involvement of MUS81 to multiple processes after replication stress.
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Replicación del ADN/genética , Proteínas de Unión al ADN/biosíntesis , Endonucleasas/biosíntesis , Proteína Recombinante y Reparadora de ADN Rad52/biosíntesis , Recombinación Genética , Puntos de Control del Ciclo Celular/genética , Supervivencia Celular/genética , Inestabilidad Cromosómica/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Regulación de la Expresión Génica , Genoma Humano , Células HEK293 , Humanos , Recombinasa Rad51/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Fase S/genéticaRESUMEN
The MUTYH DNA-glycosylase is indirectly engaged in the repair of the miscoding 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxodG) lesion by removing adenine erroneously incorporated opposite the oxidized purine. Inherited biallelic mutations in the MUTYH gene are responsible for a recessive syndrome, the MUTYH-associated polyposis (MAP), which confers an increased risk of colorectal cancer. In this study, we functionally characterized the Q338H variant using recombinant proteins, as well as cell-based assays. This is a common variant among human colorectal cancer genes, which is generally considered, unrelated to the MAP phenotype but recently indicated as a low-penetrance allele. We demonstrate that the Q338H variant retains a wild-type DNA-glycosylase activity in vitro, but it shows a reduced ability to interact with the replication sensor RAD9:RAD1:HUS1 (9-1-1) complex. In comparison with Mutyh(-)(/)(-) mouse embryo fibroblasts expressing a wild-type MUTYH cDNA, the expression of Q338H variant was associated with increased levels of DNA 8-oxodG, hypersensitivity to oxidant and accumulation of the population in the S phase of the cell cycle. Thus, an inefficient interaction of MUTYH with the 9-1-1 complex leads to a repair-defective phenotype, indicating that a proper communication between MUTYH enzymatic function and the S phase checkpoint is needed for effective repair of oxidative damage.
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ADN Glicosilasas/fisiología , Reparación del ADN , Sustitución de Aminoácidos , Animales , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Ratones , Ratones Noqueados , Mutación , Estrés Oxidativo , Proteínas Recombinantes/metabolismoRESUMEN
Accurate response to replication arrest is crucial to preserve genome stability and requires both the ATR and ATM functions. The Werner syndrome protein (WRN) is implicated in the recovery of stalled replication forks, and although an ATR/ATM-dependent phosphorylation of WRN was observed after replication arrest, the function of such modifications during the response to perturbed replication is not yet appreciated. Here, we report that WRN is directly phosphorylated by ATR at multiple C-terminal S/TQ residues. Suppression of ATR-mediated phosphorylation of WRN prevents proper accumulation of WRN in nuclear foci, co-localisation with RPA and causes breakage of stalled forks. On the other hand, inhibition of ATM kinase activity or expression of an ATM-unphosphorylable WRN allele leads to retention of WRN in nuclear foci and impaired recruitment of RAD51 recombinase resulting in reduced viability after fork collapse. Altogether, our findings indicate that ATR and ATM promote recovery from perturbed replication by differently regulating WRN at defined moments of the response to replication fork arrest.
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Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , RecQ Helicasas/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Exodesoxirribonucleasas/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , RecQ Helicasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/genética , Helicasa del Síndrome de WernerRESUMEN
The European Commission asked EFSA to update its 2011 risk assessment on tetrabromobisphenol A (TBBPA) and five derivatives in food. Neurotoxicity and carcinogenicity were considered as the critical effects of TBBPA in rodent studies. The available evidence indicates that the carcinogenicity of TBBPA occurs via non-genotoxic mechanisms. Taking into account the new data, the CONTAM Panel considered it appropriate to set a tolerable daily intake (TDI). Based on decreased interest in social interaction in male mice, a lowest observed adverse effect level (LOAEL) of 0.2 mg/kg body weight (bw) per day was identified and selected as the reference point for the risk characterisation. Applying the default uncertainty factor of 100 for inter- and intraspecies variability, and a factor of 3 to extrapolate from the LOAEL to NOAEL, a TDI for TBBPA of 0.7 µg/kg bw per day was established. Around 2100 analytical results for TBBPA in food were used to estimate dietary exposure for the European population. The most important contributors to the chronic dietary LB exposure to TBBPA were fish and seafood, meat and meat products and milk and dairy products. The exposure estimates to TBBPA were all below the TDI, including those estimated for breastfed and formula-fed infants. Accounting for the uncertainties affecting the assessment, the CONTAM Panel concluded with 90%-95% certainty that the current dietary exposure to TBBPA does not raise a health concern for any of the population groups considered. There were insufficient data on the toxicity of any of the TBBPA derivatives to derive reference points, or to allow a comparison with TBBPA that would support assignment to an assessment group for the purposes of combined risk assessment.
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The European Commission asked EFSA for a risk assessment on small organoarsenic species in food. For monomethylarsonic acid MMA(V), decreased body weight resulting from diarrhoea in rats was identified as the critical endpoint and a BMDL10 of 18.2 mg MMA(V)/kg body weight (bw) per day (equivalent to 9.7 mg As/kg bw per day) was calculated as a reference point (RP). For dimethylarsinic acid DMA(V), increased incidence in urinary bladder tumours in rats was identified as the critical endpoint. A BMDL10 of 1.1 mg DMA(V)/kg bw per day (equivalent to 0.6 mg As/kg bw per day) was calculated as an RP. For other small organoarsenic species, the toxicological data are insufficient to identify critical effects and RPs, and they could not be included in the risk assessment. For both MMA(V) and DMA(V), the toxicological database is incomplete and a margin of exposure (MOE) approach was applied for risk characterisation. The highest chronic dietary exposure to DMA(V) was estimated in 'Toddlers', with rice and fish meat as the main contributors across population groups. For MMA(V), the highest chronic dietary exposures were estimated for high consumers of fish meat and processed/preserved fish in 'Infants' and 'Elderly' age class, respectively. For MMA(V), an MOE of ≥ 500 was identified not to raise a health concern. For MMA(V), all MOEs were well above 500 for average and high consumers and thus do not raise a health concern. For DMA(V), an MOE of 10,000 was identified as of low health concern as it is genotoxic and carcinogenic, although the mechanisms of genotoxicity and its role in carcinogenicity of DMA(V) are not fully elucidated. For DMA(V), MOEs were below 10,000 in many cases across dietary surveys and age groups, in particular for some 95th percentile exposures. The Panel considers that this would raise a health concern.
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The European Commission asked EFSA to update its 2009 risk assessment on arsenic in food carrying out a hazard assessment of inorganic arsenic (iAs) and using the revised exposure assessment issued by EFSA in 2021. Epidemiological studies show that the chronic intake of iAs via diet and/or drinking water is associated with increased risk of several adverse outcomes including cancers of the skin, bladder and lung. The CONTAM Panel used the benchmark dose lower confidence limit based on a benchmark response (BMR) of 5% (relative increase of the background incidence after adjustment for confounders, BMDL05) of 0.06 µg iAs/kg bw per day obtained from a study on skin cancer as a Reference Point (RP). Inorganic As is a genotoxic carcinogen with additional epigenetic effects and the CONTAM Panel applied a margin of exposure (MOE) approach for the risk characterisation. In adults, the MOEs are low (range between 2 and 0.4 for mean consumers and between 0.9 and 0.2 at the 95th percentile exposure, respectively) and as such raise a health concern despite the uncertainties.
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EFSA was asked for a scientific opinion on the risks for animal and human health related to the presence of polychlorinated naphthalenes (PCNs) in feed and food. The assessment focused on hexaCNs due to very limited data on other PCN congeners. For hexaCNs in feed, 217 analytical results were used to estimate dietary exposures for food-producing and non-food-producing animals; however, a risk characterisation could not be performed because none of the toxicological studies allowed identification of reference points. The oral repeated dose toxicity studies performed in rats with a hexaCN mixture containing all 10 hexaCNs indicated that the critical target was the haematological system. A BMDL20 of 0.05 mg/kg body weight (bw) per day was identified for a considerable decrease in the platelet count. For hexaCNs in food, 2317 analytical results were used to estimate dietary exposures across dietary surveys and age groups. The highest exposure ranged from 0.91 to 29.8 pg/kg bw per day in general population and from 220 to 559 pg/kg bw per day for breast-fed infants with the highest consumption of breast milk. Applying a margin of exposure (MOE) approach, the estimated MOEs for the high dietary exposures ranged from 1,700,000 to 55,000,000 for the general population and from 90,000 to 230,000 for breast-fed infants with the highest consumption of breast milk. These MOEs are far above the minimum MOE of 2000 that does not raise a health concern. Taking account of the uncertainties affecting the assessment, the Panel concluded with at least 99% certainty that dietary exposure to hexaCNs does not raise a health concern for any of the population groups considered. Due to major limitations in the available data, no assessment was possible for genotoxic effects or for health risks of PCNs other than hexaCNs.
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This statement provides scientific guidance on the information needed to support the risk assessment of the detoxification processes applied to products intended for animal feed in line with the acceptability criteria of the Commission Regulation (EU) 2015/786.
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The European Commission requested EFSA to provide an update of the 2012 Scientific Opinion of the Panel on Contaminants in the Food Chain (CONTAM) on the risks for animal health related to the presence of ergot alkaloids (EAs) in feed. EAs are produced by several fungi of the Claviceps and Epichloë genera. This Opinion focussed on the 14 EAs produced by C. purpurea (ergocristine, ergotamine, ergocornine, α- and ß-ergocryptine, ergometrine, ergosine and their corresponding 'inine' epimers). Effects observed with EAs from C. africana (mainly dihydroergosine) and Epichloë (ergovaline/-inine) were also evaluated. There is limited information on toxicokinetics in food and non-food producing animals. However, transfer from feed to food of animal origin is negligible. The major effects of EAs are related to vasoconstriction and are exaggerated during extreme temperatures. In addition, EAs cause a decrease in prolactin, resulting in a reduced milk production. Based on the sum of the EAs, the Panel considered the following as Reference Points (RPs) in complete feed for adverse animal health effects: for pigs and piglets 0.6 mg/kg, for chickens for fattening and hens 2.1 and 3.7 mg/kg, respectively, for ducks 0.2 mg/kg, bovines 0.1 mg/kg and sheep 0.3 mg/kg. A total of 19,023 analytical results on EAs (only from C. purpurea) in feed materials and compound feeds were available for the exposure assessment (1580 samples). Dietary exposure was assessed using two feeding scenarios (model diets and compound feeds). Risk characterisation was done for the animals for which an RP could be identified. The CONTAM Panel considers that, based on exposure from model diets, the presence of EAs in feed raises a health concern in piglets, pigs for fattening, sows and bovines, while for chickens for fattening, laying hens, ducks, ovines and caprines, the health concern related to EAs in feed is low.
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The European Commission asked EFSA to update its 2011 risk assessment on polybrominated diphenyl ethers (PBDEs) in food, focusing on 10 congeners: BDE-28, -47, -49, -99, -100, -138, -153, -154, -183 and 209. The CONTAM Panel concluded that the neurodevelopmental effects on behaviour and reproductive/developmental effects are the critical effects in rodent studies. For four congeners (BDE-47, -99, -153, -209) the Panel derived Reference Points, i.e. benchmark doses and corresponding lower 95% confidence limits (BMDLs), for endpoint-specific benchmark responses. Since repeated exposure to PBDEs results in accumulation of these chemicals in the body, the Panel estimated the body burden at the BMDL in rodents, and the chronic intake that would lead to the same body burden in humans. For the remaining six congeners no studies were available to identify Reference Points. The Panel concluded that there is scientific basis for inclusion of all 10 congeners in a common assessment group and performed a combined risk assessment. The Panel concluded that the combined margin of exposure (MOET) approach was the most appropriate risk metric and applied a tiered approach to the risk characterisation. Over 84,000 analytical results for the 10 congeners in food were used to estimate the exposure across dietary surveys and age groups of the European population. The most important contributors to the chronic dietary Lower Bound exposure to PBDEs were meat and meat products and fish and seafood. Taking into account the uncertainties affecting the assessment, the Panel concluded that it is likely that current dietary exposure to PBDEs in the European population raises a health concern.
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Huntington disease (HD) is a neurodegenerative disease caused by expansion of CAG repeats in the huntingtin (Htt) gene. The expression of hMTH1, the human hydrolase that degrades oxidized purine nucleoside triphosphates, grants protection in a chemical HD mouse model in which HD-like features are induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). To further examine the relationship between oxidized dNTPs and HD-like neurodegeneration, we studied the effects of hMTH1 expression in a genetic cellular model for HD, such as striatal cells expressing mutant htt (Hdh(Q111)). hMTH1 expression protected these cells from 3-NP and H2O2-induced killing, by counteracting the mutant htt-dependent increased vulnerability and accumulation of nuclear and mitochondrial DNA 8-hydroxyguanine levels. hMTH1 expression reverted the decreased mitochondrial membrane potential characteristic of Hdh(Q111) cells and delayed the increase in mitochondrial reactive oxygen species associated with 3-NP treatment. Further indications of hMTH1-mediated mitochondrial protection are the partial reversion of 3-NP-induced alterations in mitochondrial morphology and the modulation of DRP1 and MFN1 proteins, which control fusion/fission rates of mitochondria. Finally, in line with the in vitro findings, upon 3-NP in vivo treatment, 8-hydroxyguanine levels in mitochondrial DNA from heart, muscle and brain are significantly lower in transgenic hMTH1-expressing mice than in wild-type animals.
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Enzimas Reparadoras del ADN/metabolismo , Enfermedad de Huntington/fisiopatología , Mitocondrias/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Encéfalo/patología , Encéfalo/fisiopatología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular , Células Cultivadas , ADN Mitocondrial/metabolismo , Modelos Animales de Enfermedad , Humanos , Proteína Huntingtina , Enfermedad de Huntington/patología , Peróxido de Hidrógeno/toxicidad , Potencial de la Membrana Mitocondrial/fisiología , Ratones Transgénicos , Mitocondrias/patología , Músculo Esquelético/metabolismo , Mutación , Miocardio/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nitrocompuestos/toxicidad , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Propionatos/toxicidad , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Retinitis pigmentosa (RP) is a genetically heterogenous group of inherited retinal degenerative diseases resulting from photoreceptor cell death and affecting >1 million persons globally. Although oxidative stress has been implicated in the pathogenesis of RP, the mechanisms by which oxidative stress mediates photoreceptor cell death are largely unknown. Here, we show that oxidation of nucleic acids is a key component in the initiation of death-signaling pathways in rd10 mice, a model of RP. Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) increased in photoreceptor cells, and especially within their nuclei, in rd10 mice as well as in Royal College of Surgeons rats, another model of RP caused by different genetic mutations. Vitreous samples from humans with RP contained higher levels of 8-oxo-dG excreted than samples from nondegenerative controls. Transgenic overexpression of human MutT homolog-1, which hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, significantly attenuated 8-oxo-dG accumulation in nuclear DNA and photoreceptor cell death in rd10 mice, in addition to suppressing DNA single-strand break formation, poly(ADP-ribose) polymerase activation, and nuclear translocation of apoptosis-inducing factor. These findings indicate that oxidative DNA damage is an important process for the triggering of photoreceptor cell death in rd10 mice and suggest that stimulation of DNA repair enzymes may be a novel therapeutic approach to attenuate photoreceptor cell loss in RP.
Asunto(s)
Daño del ADN , Enzimas Reparadoras del ADN/metabolismo , Patrón de Herencia/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Animales , Factor Inductor de la Apoptosis/metabolismo , Calpaína/metabolismo , Muerte Celular , Núcleo Celular/metabolismo , Roturas del ADN de Cadena Simple , Modelos Animales de Enfermedad , Activación Enzimática , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxidación-Reducción , Poli Adenosina Difosfato Ribosa/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transporte de Proteínas , Ratas , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Transducción de SeñalRESUMEN
MUTYH, a human ortholog of MutY, is a post-replicative DNA glycosylase, highly conserved throughout evolution, involved in the correction of mismatches resulting from a faulty replication of the oxidized base 8-hydroxyguanine (8-oxodG). In particular removal of adenine from A:8-oxodG mispairs by MUTYH activity is followed by error-free base excision repair (BER) events, leading to the formation of C:8-oxodG base pairs. These are the substrate of another BER enzyme, the OGG1 DNA glycosylase, which then removes 8-oxodG from DNA. Thus the combined action of OGG1 and MUTYH prevents oxidative damage-induced mutations, i.e. GC->TA transversions. Germline mutations in MUTYH are associated with a recessively heritable colorectal polyposis, now referred to as MUTYH-associated polyposis (MAP). Here we will review the phenotype(s) associated with MUTYH inactivation from bacteria to mammals, the structure of the MUTYH protein, the molecular mechanisms of its enzymatic activity and the functional characterization of MUTYH variants. The relevance of these results will be discussed to define the role of specific human mutations in colorectal cancer risk together with the possible role of MUTYH inactivation in sporadic cancer.