RÉSUMÉ
Increased risk for the development of hepatocellular carcinoma (HCC) is driven by a number of etiological factors including hepatitis viral infection and dietary exposures to foods contaminated with aflatoxin-producing molds. Intracellular metabolic activation of aflatoxin B1 (AFB1) to a reactive epoxide generates highly mutagenic AFB1-Fapy-dG adducts. Previously, we demonstrated that repair of AFB1-Fapy-dG adducts can be initiated by the DNA glycosylase NEIL1 and that male Neil1-/- mice were significantly more susceptible to AFB1-induced HCC relative to wild-type mice. To investigate the mechanisms underlying this enhanced carcinogenesis, WT and Neil1-/- mice were challenged with a single, 4 mg/kg dose of AFB1 and frequencies and spectra of mutations were analyzed in liver DNAs 2.5 months post-injection using duplex sequencing. The analyses of DNAs from AFB1-challenged mice revealed highly elevated mutation frequencies in the nuclear genomes of both males and females, but not the mitochondrial genomes. In both WT and Neil1-/- mice, mutation spectra were highly similar to the AFB1-specific COSMIC signature SBS24. Relative to wild-type, the NEIL1 deficiency increased AFB1-induced mutagenesis with concomitant elevated HCCs in male Neil1-/- mice. Our data establish a critical role of NEIL1 in limiting AFB1-induced mutagenesis and ultimately carcinogenesis.
RÉSUMÉ
Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G â T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 µg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.
Sujet(s)
Aflatoxine B1 , Adduits à l'ADN , Souris , Humains , Animaux , Aflatoxine B1/composition chimique , Inclusion en paraffine , ADN/métabolisme , Cancérogènes/métabolisme , Spectrométrie de masse , Guanine , FormaldéhydeRÉSUMÉ
Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1gbw/bw null mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell's kinocilial links. Consistent with other Usher type 1 models, Ush1gbw/bw mice had no substantial retinal degeneration compared with Ush1gbw /+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene.
Sujet(s)
DNA Glycosylases , Syndromes d'Usher , Souris , Animaux , Allèles , Syndromes d'Usher/génétique , Mutation , Phénotype , DNA Glycosylases/génétiqueRÉSUMÉ
Aflatoxin B1 (AFB1) exposure through contaminated food is a primary contributor to hepatocellular carcinogenesis worldwide. Hepatitis B viral infections in livers dramatically increase the carcinogenic potency of AFB1 exposures. Liver cytochrome P450 oxidizes AFB1 to the epoxide, which in turn reacts with N7-guanine in DNA, producing the cationic trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct (AFB1-N7-Gua). The opening of the imidazole ring of AFB1-N7-Gua under physiological conditions causes the formation of the cis- and trans-diastereomers of 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua). These adducts primarily lead to G â T mutations, with AFB1-FapyGua being significantly more mutagenic than AFB1-N7-Gua. The unequivocal identification and accurate quantification of these AFB1-Gua adducts as biomarkers are essential for a fundamental understanding and prevention of AFB1-induced hepatocellular carcinogenesis. Among a variety of analytical techniques used for this purpose, liquid chromatography-tandem mass spectrometry, with the use of the stable isotope-labeled analogues of AFB1-FapyGua and AFB1-N7-Gua as internal standards, provides the greatest accuracy and sensitivity. cis-AFB1-FapyGua-15N5, trans-AFB1-FapyGua-15N5, and AFB1-N7-Gua-15N5 have been synthesized and used successfully as internal standards. However, the availability of these standards from either academic institutions or commercial sources ceased to exist. Thus, quantitative genomic data regarding AFB1-induced DNA damage in animal models and humans remain challenging to obtain. Previously, AFB1-N7-Gua-15N5 was prepared by reacting AFB1-exo-8,9-epoxide with the uniformly 15N5-labeled DNA isolated from algae grown in a pure 15N-environment, followed by alkali treatment, resulting in the conversion of AFB1-N7-Gua-15N5 to AFB1-FapyGua-15N5. In the present work, we used a different and simpler approach to synthesize cis-AFB1-FapyGua-15N5, trans-AFB1-FapyGua-15N5, and AFB1-N7-Gua-15N5 from a partial double-stranded 11-mer Gua-15N5-labeled oligodeoxynucleotide, followed by isolation and purification. We also show the validation of these 15N5-labeled standards for the measurement of cis-AFB1-FapyGua, trans-AFB1-FapyGua, and AFB1-N7-Gua in DNA of livers of AFB1-treated mice.
RÉSUMÉ
Obesity and related metabolic disorders are pressing public health concerns, raising the risk for a multitude of chronic diseases. Obesity is multi-factorial disease, with both diet and lifestyle, as well as genetic and developmental factors leading to alterations in energy balance. In this regard, a novel role for DNA repair glycosylases in modulating risk for obesity has been previously established. Global deletion of either of two different glycosylases with varying substrate specificities, Nei-like endonuclease 1 (NEIL1) or 8-oxoguanine DNA glycosylase-1 (OGG1), both predispose mice to diet-induced obesity (DIO). Conversely, enhanced expression of the human OGG1 gene renders mice resistant to obesity and adiposity. This resistance to DIO is mediated through increases in whole body energy expenditure and increased respiration in adipose tissue. Here, we report that hOGG1 expression also confers resistance to genetically-induced obesity. While Agouti obese (Ay/a) mice are hyperphagic and consequently develop obesity on a chow diet, hOGG1 expression in Ay/a mice (Ay/aTg ) prevents increased body weight, without reducing food intake. Instead, obesity resistance in Ay/aTg mice is accompanied by increased whole body energy expenditure and tissue mitochondrial content. We also report for the first time that OGG1-mediated obesity resistance in both the Ay/a model and DIO model requires maternal transmission of the hOGG1 transgene. Maternal, but not paternal, transmission of the hOGG1 transgene is associated with obesity resistance and increased mitochondrial content in adipose tissue. These data demonstrate a critical role for OGG1 in modulating energy balance through changes in adipose tissue function. They also demonstrate the importance of OGG1 in modulating developmental programming of mitochondrial content and quality, thereby determining metabolic outcomes in offspring.
RÉSUMÉ
OGG1-deficient (Ogg1-/-) animals display increased propensity to age-induced and diet-induced metabolic diseases, including insulin resistance and fatty liver. Since the intestinal microbiome is increasingly understood to play a role in modulating host metabolic responses, we examined gut microbial composition in Ogg1-/- mice subjected to different nutritional challenges. Interestingly, Ogg1-/- mice had a markedly altered intestinal microbiome under both control-fed and hypercaloric diet conditions. Several microbial species that were increased in Ogg1-/- animals were associated with increased energy harvest, consistent with their propensity to high-fat diet induced weight gain. In addition, several pro-inflammatory microbes were increased in Ogg1-/- mice. Consistent with this observation, Ogg1-/- mice were significantly more sensitive to intestinal inflammation induced by acute exposure to dextran sulfate sodium. Taken together, these data indicate that in addition to their proclivity to obesity and metabolic disease, Ogg1-/- mice are prone to colonic inflammation. Further, these data point to alterations in the intestinal microbiome as potential mediators of the metabolic and intestinal inflammatory response in Ogg1-/- mice.
Sujet(s)
DNA Glycosylases/génétique , Microbiome gastro-intestinal , Animaux , Bacteroidetes/isolement et purification , Biodiversité , Poids , Colite/induit chimiquement , Colite/anatomopathologie , DNA Glycosylases/déficit , Sulfate dextran/toxicité , Alimentation riche en graisse , Modèles animaux de maladie humaine , Métabolisme énergétique , Firmicutes/isolement et purification , Génotype , Mâle , Maladies métaboliques/métabolisme , Maladies métaboliques/anatomopathologie , Souris , Souris de lignée C57BL , Souris knockout , Obésité/métabolisme , Obésité/anatomopathologie , Analyse en composantes principalesRÉSUMÉ
Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid change of lysine 242 to arginine. Previous investigations of the catalytic efficiencies of the two forms of the enzyme revealed differential release of thymine glycol (ThyGly) from synthetic oligodeoxynucleotides, with the unedited form, NEIL1 K242 being ≈30-fold more efficient than the edited NEIL1 K242R. In contrast, when these enzymes were reacted with oligodeoxynucleotides containing guanidinohydantoin or spiroiminohydantoin, the edited K242R form was ≈3-fold more efficient than the unedited NEIL1. However, no prior studies have investigated the efficiencies of these two forms of NEIL1 on either high-molecular weight DNA containing multiple oxidatively-induced base damages, or oligodeoxynucleotides containing a bulky alkylated formamidopyrimidine. To understand the extent of changes in substrate recognition, γ-irradiated calf thymus DNA was treated with either edited or unedited NEIL1 and the released DNA base lesions analyzed by gas chromatography-tandem mass spectrometry. Of all the measured DNA lesions, imidazole ring-opened 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were preferentially released by both NEIL1 enzymes with K242R being ≈1.3 and 1.2-fold more efficient than K242 on excision of FapyAde and FapyGua, respectively. Consistent with the prior literature, large differences (≈7.5 to 12-fold) were measured in the excision of ThyGly from genomic DNA by the unedited versus edited NEIL1. In contrast, the edited NEIL1 was more efficient (≈3 to 5-fold) on release of 5-hydroxycytosine. Excision kinetics on DNA containing a site-specific aflatoxin B1-FapyGua adduct revealed an ≈1.4-fold higher rate by the unedited NEIL1. Molecular modeling provides insight into these differential substrate specificities. The results of this study and in particular, the comparison of substrate specificities of unedited and edited NEIL1 using biologically and clinically important base lesions, are critical for defining its role in preservation of genomic integrity.
Sujet(s)
Adenosine deaminase/métabolisme , Substitution d'acide aminé , Adduits à l'ADN/métabolisme , DNA Glycosylases/métabolisme , Protéines de liaison à l'ARN/métabolisme , Domaine catalytique , DNA Glycosylases/composition chimique , DNA Glycosylases/génétique , Chromatographie gazeuse-spectrométrie de masse , Édition de gène , Humains , Modèles moléculaires , Masse moléculaire , Conformation des protéines , Spécificité du substratRÉSUMÉ
The combination of chronic dietary exposure to the fungal toxin, aflatoxin B1 (AFB1), and hepatitis B viral (HBV) infection is associated with an increased risk for early onset hepatocellular carcinomas (HCCs). An in-depth knowledge of the mechanisms driving carcinogenesis is critical for the identification of genetic risk factors affecting the susceptibility of individuals who are HBV infected and AFB1 exposed. AFB1-induced mutagenesis is characterized by G to T transversions. Hence, the DNA repair pathways that function on AFB1-induced DNA adducts or base damage from HBV-induced inflammation are anticipated to have a strong role in limiting carcinogenesis. These pathways define the mutagenic burden in the target tissues and ultimately limit cellular progression to cancer. Murine data have demonstrated that NEIL1 in the DNA base excision repair pathway was significantly more important than nucleotide excision repair relative to elevated risk for induction of HCCs. These data suggest that deficiencies in NEIL1 could contribute to the initiation of HCCs in humans. To investigate this hypothesis, publicly-available data on variant alleles of NEIL1 were analyzed and compared with genome sequencing data from HCC tissues derived from individuals residing in Qidong County (China). Three variant alleles were identified and the corresponding A51V, P68H, and G245R enzymes were characterized for glycosylase activity on genomic DNA containing a spectrum of oxidatively-induced base damage and an oligodeoxynucleotide containing a site-specific AFB1-formamidopyrimidine guanine adduct. Although the efficiency of the P68H variant was modestly decreased, the A51V and G245R variants showed nearly wild-type activities. Consistent with biochemical findings, molecular modeling of these variants demonstrated only slight local structural alterations. However, A51V was highly temperature sensitive suggesting that its biological activity would be greatly reduced. Overall, these studies have direct human health relevance pertaining to genetic risk factors and biochemical pathways previously not recognized as germane to induction of HCCs.
Sujet(s)
DNA Glycosylases/génétique , Réparation de l'ADN , Mutation , Polymorphisme de nucléotide simple , Asiatiques/génétique , Adduits à l'ADN , DNA Glycosylases/composition chimique , DNA Glycosylases/métabolisme , Stabilité enzymatique , Escherichia coli , Humains , Domaines protéiques , Spécificité du substratRÉSUMÉ
Dietary exposure to aflatoxin B1 (AFB1) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB1 exposure leads to the formation of AFB1-N7-guanine (AFB1-N7-Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) in DNA. These adducts lead to G â T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB1 as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB1-N7-Gua and the two diastereomers of AFB1-FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB1-treated mice. Levels varying from 1.5 to 45 lesions/106 DNA bases in AFB1-treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB1 treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB1 treatment of mice. On the other hand, significantly increased levels of (5' R)- and (5' S)-8,5'-cyclo-2'-deoxyadenosines were observed in liver DNA of AFB1-treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB1-DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB1 exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.
Sujet(s)
Aflatoxines/composition chimique , Aflatoxines/pharmacologie , Adduits à l'ADN/composition chimique , Altération de l'ADN , Guanine/composition chimique , Technique de dilution radioisotopique , Aflatoxines/administration et posologie , Animaux , Chromatographie en phase liquide , Foie/effets des médicaments et des substances chimiques , Foie/métabolisme , Souris , Souris de lignée C57BL , Souris knockout , Conformation moléculaire , OxydoréductionRÉSUMÉ
Obesity and related metabolic pathologies represent a significant public health concern. Obesity is associated with increased oxidative stress that damages genomic and mitochondrial DNA. Oxidatively-induced lesions in both DNA pools are repaired via the base-excision repair pathway, initiated by DNA glycosylases such as 8-oxoguanine DNA glycosylase (OGG1). Global deletion of OGG1 and common OGG1 polymorphisms render mice and humans susceptible to metabolic disease. However, the relative contribution of mitochondrial OGG1 to this metabolic phenotype is unknown. Here, we demonstrate that transgenic targeting of OGG1 to mitochondria confers significant protection from diet-induced obesity, insulin resistance, and adipose tissue inflammation. These favorable metabolic phenotypes are mediated by an increase in whole body energy expenditure driven by specific metabolic adaptations, including increased mitochondrial respiration in white adipose tissue of OGG1 transgenic (Ogg1Tg) animals. These data demonstrate a critical role for a DNA repair protein in modulating mitochondrial energetics and whole-body energy balance.
Sujet(s)
Tissu adipeux blanc/métabolisme , DNA Glycosylases/métabolisme , Métabolisme énergétique , Mitochondries/métabolisme , Obésité/métabolisme , Animaux , DNA Glycosylases/génétique , Réparation de l'ADN , Délétion de gène , Ciblage de gène , Souris de lignée C57BL , Mitochondries/génétique , Obésité/étiologie , Obésité/génétique , Facteurs de protectionRÉSUMÉ
The molecular basis for ultraviolet (UV) light-induced nonmelanoma and melanoma skin cancers centers on cumulative genomic instability caused by inefficient DNA repair of dipyrimidine photoproducts. Inefficient DNA repair and subsequent translesion replication past these DNA lesions generate distinct molecular signatures of tandem CC to TT and C to T transitions at dipyrimidine sites. Since previous efforts to develop experimental strategies to enhance the repair capacity of basal keratinocytes have been limited, we have engineered the N-terminally truncated form (Δ228) UV endonuclease (UVDE) from Schizosaccharomyces pombe to include a TAT cell-penetrating peptide sequence with or without a nuclear localization signal (NLS): UVDE-TAT and UVDE-NLS-TAT. Further, a NLS was engineered onto a pyrimidine dimer glycosylase from Paramecium bursaria chlorella virus-1 (cv-pdg-NLS). Purified enzymes were encapsulated into liposomes and topically delivered to the dorsal surface of SKH1 hairless mice in a UVB-induced carcinogenesis study. Total tumor burden was significantly reduced in mice receiving either UVDE-TAT or UVDE-NLS-TAT versus control empty liposomes and time to death was significantly reduced with the UVDE-NLS-TAT. These data suggest that efficient delivery of exogenous enzymes for the initiation of repair of UVB-induced DNA damage may protect from UVB induction of squamous and basal cell carcinomas.
Sujet(s)
Carcinogenèse/effets des radiations , Réparation de l'ADN , Tumeurs cutanées/prévention et contrôle , Rayons ultraviolets , Animaux , Enzymes de réparation de l'ADN/administration et posologie , Enzymes de réparation de l'ADN/génétique , Enzymes de réparation de l'ADN/métabolisme , Souris hairless , Protéines recombinantes/administration et posologie , Protéines recombinantes/génétique , Protéines recombinantes/métabolismeRÉSUMÉ
Oxidative stress resulting from endogenous and exogenous sources causes damage to cellular components, including genomic and mitochondrial DNA. Oxidative DNA damage is primarily repaired via the base excision repair pathway that is initiated by DNA glycosylases. 8-oxoguanine DNA glycosylase (OGG1) recognizes and cleaves oxidized and ring-fragmented purines, including 8-oxoguanine, the most commonly formed oxidative DNA lesion. Mice lacking the OGG1 gene product are prone to multiple features of the metabolic syndrome, including high-fat diet-induced obesity, hepatic steatosis, and insulin resistance. Here, we report that OGG1-deficient mice also display skeletal muscle pathologies, including increased muscle lipid deposition and alterations in genes regulating lipid uptake and mitochondrial fission in skeletal muscle. In addition, expression of genes of the TCA cycle and of carbohydrate and lipid metabolism are also significantly altered in muscle of OGG1-deficient mice. These tissue changes are accompanied by marked reductions in markers of muscle function in OGG1-deficient animals, including decreased grip strength and treadmill endurance. Collectively, these data indicate a role for skeletal muscle OGG1 in the maintenance of optimal tissue function.
Sujet(s)
DNA Glycosylases/déficit , Métabolisme lipidique/physiologie , Mitochondries/métabolisme , Muscles squelettiques/métabolisme , Adiposité , Animaux , Altération de l'ADN/physiologie , DNA Glycosylases/génétique , ADN mitochondrial/métabolisme , Acides gras/métabolisme , Analyse de profil d'expression de gènes , Mâle , Souris de lignée C57BL , Mitochondries/génétique , Dynamique mitochondriale/physiologie , Modèles animaux , Force musculaire/physiologie , Muscles squelettiques/anatomopathologie , Endurance physique/physiologie , Acide pyruvique/métabolisme , Course à pied/physiologieRÉSUMÉ
Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB1) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB1-induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB1-deoxyguanosine adduct (AFB1-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB1 show significant increases in the levels of AFB1-Fapy-dG in Neil1-/- vs. wild-type liver DNA. Further, Neil1-/- mice are highly susceptible to AFB1-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1-/- The magnitude of this effect in Neil1-/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB1-associated HCCs.
Sujet(s)
Aflatoxines/toxicité , Carcinome hépatocellulaire/prévention et contrôle , Adduits à l'ADN/effets des médicaments et des substances chimiques , DNA Glycosylases/physiologie , Tumeurs expérimentales du foie/prévention et contrôle , Agents protecteurs/pharmacologie , Animaux , Carcinome hépatocellulaire/induit chimiquement , Carcinome hépatocellulaire/métabolisme , Carcinome hépatocellulaire/anatomopathologie , Femelle , Tumeurs expérimentales du foie/induit chimiquement , Tumeurs expérimentales du foie/métabolisme , Tumeurs expérimentales du foie/anatomopathologie , Mâle , Souris , Souris de lignée C57BL , Souris knockout , Toxiques/toxicitéRÉSUMÉ
Oxidative stress and reactive oxygen species (ROS)-induced DNA base damage are thought to be central mediators of UV-induced carcinogenesis and skin aging. However, increased steady-state levels of ROS-induced DNA base damage have not been reported after chronic UV exposure. Accumulation of ROS-induced DNA base damage is governed by rates of lesion formation and repair. Repair is generally performed by Base Excision Repair (BER), which is initiated by DNA glycosylases, such as 8-oxoguanine glycosylase and Nei-Endonuclease VIII-Like 1 (NEIL1). In the current study, UV light (UVB) was used to elicit protracted low-level ROS challenge in wild-type (WT) and Neil1-/- mouse skin. Relative to WT controls, Neil1-/- mice showed an increased sensitivity to tissue destruction from the chronic UVB exposure, and corresponding enhanced chronic inflammatory responses as measured by cytokine message levels and profiling, as well as neutrophil infiltration. Additionally, levels of several ROS-induced DNA lesions were measured including 4,6-diamino-5-formamidopyrimidine (FapyGua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyAde), 8-hydroxyguanine (8-OH-Gua), 5,6-dihydroxyuracil (5,6-diOH-Ura) and thymine glycol (ThyGly). In WT mice, chronic UVB exposure led to increased steady-state levels of FapyGua, FapyAde, and ThyGly with no significant increases in 8-OH-Gua or 5,6-diOH-Ura. Interestingly, the lesions that accumulated were all substrates of NEIL1. Collectively, these data suggest that NEIL1-initiated repair of a subset of ROS-induced DNA base lesions may be insufficient to prevent the initiation of inflammatory pathways during chronic UV exposure in mouse skin.
Sujet(s)
DNA Glycosylases/génétique , Réparation de l'ADN , Espèces réactives de l'oxygène/métabolisme , Peau/effets des radiations , Animaux , Cytokines/biosynthèse , Cytokines/génétique , Altération de l'ADN , DNA Glycosylases/déficit , DNA Glycosylases/métabolisme , Analyse de profil d'expression de gènes , Régulation de l'expression des gènes , Guanine/analogues et dérivés , Guanine/métabolisme , Inflammation/étiologie , Inflammation/métabolisme , Inflammation/anatomopathologie , Souris , Souris knockout , Infiltration par les neutrophiles/effets des radiations , Stress oxydatif , Pyrimidines/métabolisme , Espèces réactives de l'oxygène/agonistes , Peau/métabolisme , Peau/anatomopathologie , Thymine/analogues et dérivés , Thymine/métabolisme , Rayons ultraviolets , Uracile/analogues et dérivés , Uracile/métabolismeRÉSUMÉ
Oxidative damage to DNA is mainly repaired via base excision repair, a pathway that is catalyzed by DNA glycosylases such as 8-oxoguanine DNA glycosylase (OGG1). While OGG1 has been implicated in maintaining genomic integrity and preventing tumorigenesis, we report a novel role for OGG1 in altering cellular and whole body energy homeostasis. OGG1-deficient (Ogg1(-/-)) mice have increased adiposity and hepatic steatosis following exposure to a high-fat diet (HFD), compared to wild-type (WT) animals. Ogg1(-/-) animals also have higher plasma insulin levels and impaired glucose tolerance upon HFD feeding, relative to WT counterparts. Analysis of energy expenditure revealed that HFD-fed Ogg1(-/-) mice have a higher resting VCO(2) and consequently, an increased respiratory quotient during the resting phase, indicating a preference for carbohydrate metabolism over fat oxidation in these mice. Additionally, microarray and quantitative PCR analyses revealed that key genes of fatty acid oxidation, including carnitine palmitoyl transferase-1, and the integral transcriptional co-activator Pgc-1α were significantly downregulated in Ogg1(-/-) livers. Multiple genes involved in TCA cycle metabolism were also significantly reduced in livers of Ogg1(-/-) mice. Furthermore, hepatic glycogen stores were diminished, and fasting plasma ketones were significantly reduced in Ogg1(-/-) mice. Collectively, these data indicate that OGG1 deficiency alters cellular substrate metabolism, favoring a fat sparing phenotype, that results in increased susceptibility to obesity and related pathologies in Ogg1(-/-) mice.
Sujet(s)
Adiposité , DNA Glycosylases/physiologie , Alimentation riche en graisse/effets indésirables , Stéatose hépatique/étiologie , Obésité/étiologie , Animaux , ADN mitochondrial/génétique , Métabolisme énergétique , Stéatose hépatique/métabolisme , Stéatose hépatique/anatomopathologie , Glucose/métabolisme , Hyperglycémie provoquée , Glycogène/métabolisme , Insuline/métabolisme , Lipides/analyse , Mâle , Souris , Souris de lignée C57BL , Souris knockout , Obésité/métabolisme , Obésité/anatomopathologie , Stress oxydatifRÉSUMÉ
Exposure to chronic and acute oxidative stress is correlated with many human diseases, including, but not limited to, cancer, heart disease, diabetes, and obesity. In addition to cellular lipids and proteins, cellular oxidative stress can result in damage to DNA bases, especially in mitochondrial DNA. We previously described the development of spontaneous late-onset obesity, hepatic steatosis, hyperinsulinemia, and hyperleptinemia in mice that are deficient in the DNA glycosylase nei-like 1 (NEIL1), which initiates base excision repair of several oxidatively damaged bases. In the current study, we report that exposure to a chronic oxidative stress in the form of a high-fat diet greatly accelerates the development of obesity in neil1(-/-) mice. Following a 5-wk high-fat diet challenge, neil1(-/-) mice gained significantly more body weight than neil1(+/+) littermates and had increased body fat accumulation and moderate to severe hepatic steatosis. Analysis of oxygen consumption by indirect calorimetry indicated a modest reduction in total oxygen consumption in neil1(-/-) mice that was abolished upon correction for lean body mass. Additionally, hepatic expression of several inflammatory genes was significantly upregulated in neil1(-/-) mice following high-fat diet challenge compared with chow-fed or neil1(+/+) counterparts. A long-term high-fat diet also induced glucose intolerance as well as a significant reduction in mitochondrial DNA and protein content in neil1(-/-) mice. Collectively, these data indicate that NEIL1 deficiency results in an increased susceptibility to obesity and related complications potentially by lowering the threshold for tolerance of cellular oxidative stress in neil1(-/-) mice.
Sujet(s)
Adiposité , DNA Glycosylases/génétique , Obésité/génétique , Obésité/métabolisme , Pénétrance , Adiposité/effets des médicaments et des substances chimiques , Adiposité/génétique , Animaux , Régime athérogène , Matières grasses alimentaires/effets indésirables , Femelle , Prédisposition génétique à une maladie , Mâle , Souris , Souris de lignée C57BL , Souris knockout , Obésité/épidémiologie , Obésité/étiologie , Stress oxydatif/génétique , Stress oxydatif/physiologie , Phénotype , Prévalence , Études de validation comme sujetRÉSUMÉ
The DNA repair enzyme NEIL1 is a DNA glycosylase that is involved in the first step of base excision repair (BER) of oxidatively induced DNA damage. NEIL1 exhibits a strong preference for excision of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA with no specificity for 8-hydroxyguanine (8-OH-Gua). In this study, we report on the significant accumulation of (5'R)-8,5'-cyclo-2'-deoxyadenosine (R-cdA) and (5'S)-8,5'-cyclo-2'-deoxyadenosine (S-cdA) in liver DNA of neil1(-/-) mice that were not exposed to exogenous oxidative stress, while no accumulation of these lesions was observed in liver DNA from control or ogg1(-/-) mice. Significant accumulation of FapyGua was detected in liver DNA of both neil1(-/-) and ogg1(-/-) mice, while 8-OH-Gua accumulated in ogg1(-/-) only. Since R-cdA and S-cdA contain an 8,5'-covalent bond between the base and sugar moieties, they cannot be repaired by BER. There is evidence that these lesions are repaired by nucleotide excision repair (NER). Since the accumulation of R-cdA and S-cdA in neil1(-/-) mice strongly points to the failure of their repair, these data suggest that NEIL1 is involved in NER of R-cdA and S-cdA. Further studies aimed at elucidating the mechanism of action of NEIL1 in NER are warranted.
Sujet(s)
DNA Glycosylases/physiologie , Réparation de l'ADN , Désoxyadénosine/métabolisme , Animaux , Altération de l'ADN/génétique , DNA Glycosylases/déficit , DNA Glycosylases/génétique , Réparation de l'ADN/génétique , Réplication de l'ADN/génétique , Désoxyadénosine/génétique , Guanine/analogues et dérivés , Guanine/métabolisme , Foie/composition chimique , Foie/métabolisme , Souris , Souris knockout , Pyrimidines/métabolisme , Stéréoisomérie , Spécificité du substrat/génétiqueRÉSUMÉ
We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1-/-Neil1-/- mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1-/- and Neil1-/-. The pulmonary tumors contained, exclusively, activating GGT-->GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT-->GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1-/- and Neil1-/-Nth1-/- mice. The high incidence of tumors in our Nth1-/-Neil1-/- mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua.
Sujet(s)
Altération de l'ADN , DNA Glycosylases/métabolisme , Deoxyribonuclease (pyrimidine dimer)/métabolisme , Délétion de gène , Animaux , Séquence nucléotidique , Encéphale/métabolisme , Encéphale/anatomopathologie , DNA Glycosylases/génétique , Analyse de mutations d'ADN , Deoxyribonuclease (pyrimidine dimer)/génétique , Femelle , Chromatographie gazeuse-spectrométrie de masse , Gènes ras/génétique , Guanine/analogues et dérivés , Guanine/métabolisme , Rein/métabolisme , Rein/anatomopathologie , Foie/métabolisme , Foie/anatomopathologie , Tumeurs du foie/génétique , Tumeurs du foie/métabolisme , Tumeurs du foie/anatomopathologie , Tumeurs du poumon/génétique , Tumeurs du poumon/métabolisme , Tumeurs du poumon/anatomopathologie , Mâle , Souris , Souris de lignée C57BL , Lignées consanguines de souris , Souris knockout , Mutation , Oxydoréduction , Pyrimidines/métabolismeRÉSUMÉ
Endogenously formed reactive oxygen species continuously damage cellular constituents including DNA. These challenges, coupled with exogenous exposure to agents that generate reactive oxygen species, are both associated with normal aging processes and linked to cardiovascular disease, cancer, cataract formation, and fatty liver disease. Although not all of these diseases have been definitively shown to originate from mutations in nuclear DNA or mitochondrial DNA, repair of oxidized, saturated, and ring-fragmented bases via the base excision repair pathway is known to be critical for maintaining genomic stability. One enzyme that initiates base excision repair of ring-fragmented purines and some saturated pyrimidines is NEIL1, a mammalian homolog to Escherichia coli endonuclease VIII. To investigate the organismal consequences of a deficiency in NEIL1, a knockout mouse model was created. In the absence of exogenous oxidative stress, neil1 knockout (neil1-/-) and heterozygotic (neil1+/-) mice develop severe obesity, dyslipidemia, and fatty liver disease and also have a tendency to develop hyperinsulinemia. In humans, this combination of clinical manifestations, including hypertension, is known as the metabolic syndrome and is estimated to affect >40 million people in the United States. Additionally, mitochondrial DNA from neil1-/- mice show increased levels of steady-state DNA damage and deletions relative to wild-type controls. These data suggest an important role for NEIL1 in the prevention of the diseases associated with the metabolic syndrome.