RESUMO
Micronuclei (MN) are induced by various genotoxic stressors and amass nuclear- and cytoplasmic-resident proteins, priming the cell for MN-driven signaling cascades. Here, we measured the proteome of micronuclear, cytoplasmic, and nuclear fractions from human cells exposed to a panel of six genotoxins, comprehensively profiling their MN protein landscape. We find that MN assemble a proteome distinct from both surrounding cytoplasm and parental nuclei, depleted of spliceosome and DNA damage repair components while enriched for a subset of the replisome. We show that the depletion of splicing machinery within transcriptionally active MN contributes to intra-MN DNA damage, a known precursor to chromothripsis. The presence of transcription machinery in MN is stress-dependent, causing a contextual induction of MN DNA damage through spliceosome deficiency. This dataset represents a unique resource detailing the global proteome of MN, guiding mechanistic studies of MN generation and MN-associated outcomes of genotoxic stress.
Assuntos
Cromotripsia , Proteoma , Humanos , Proteoma/genética , Proteoma/metabolismo , Proteômica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Dano ao DNA/genéticaRESUMO
The biosynthetic machinery for the production of colibactin is encoded by 19 genes (clbA - S) within the pks pathogenicity island harboured by many E. coli of the B2-phylogroup. Colibactin is a potent genotoxic metabolite which causes DNA-damage and which has potential roles in microbial competition and fitness of pks+ bacteria. Colibactin has also been strongly implicated in the development of colorectal cancer. Given the genotoxicity of colibactin and the metabolic cost of its synthesis, the regulatory system governing the clb cluster is accordingly highly complex, and many of the mechanisms remain to be elucidated. In this review we summarise the current understanding of regulation of colibactin biosynthesis by internal molecular components and how these factors are modulated by signals from the external environment.
Assuntos
Proteínas de Escherichia coli , Policetídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas de Escherichia coli/metabolismo , Policetídeos/metabolismoRESUMO
BACKGROUND: New evidence suggests that bacteria-produced DNA toxins may have a role in the development or progression of prostate cancer. To determine the prevalence of these genes in a noninfection (i.e., colonized) state, we screened urine specimens in men before undergoing a biopsy for prostate cancer detection. METHODS: We developed a multiplex polymerase chain reaction using three of the most described bacterial genotoxin gene primers: Colibactin (polyketone synthase [pks] gene island: clbN and clbB), cytotoxic necrotizing factor (cnf1) toxin, and cytolethal distending toxin B (cdtB) represented gene islands. After calibration on Escherichia coli samples of known genotypes, we used a training and validation cohort. We performed multiplex testing on a training cohort of previously collected urine from 45 men undergoing prostate biopsy. For the validation cohort, we utilized baseline urine samples from a previous randomized clinical trial (n = 263) with known prostate cancer outcomes. RESULTS: The prevalence of four common bacterial genotoxin genes detected in the urine before prostate biopsy for prostate cancer is 8% (25/311). The prevalence of pks island (clbN and clbB), cnf1, and cdt toxin genes are 6.1%, 2.4%, and 1.7%, respectively. We found no association between urinary genotoxins and prostate cancer (p = 0.83). We did identify a higher proportion of low-grade cancer (92% vs. 44%) in those men positive for urinary genotoxin and higher-grade cancer in those genotoxin negative (8% vs. 56%, p = 0.001). CONCLUSIONS: The prevalence of urinary genotoxins is low and does not correspond to a prostate cancer diagnosis. The urine was taken at one point in time and does not rule out the possibility of previous exposure.
Assuntos
Escherichia coli , Neoplasias da Próstata , Masculino , Humanos , Prevalência , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/epidemiologia , Neoplasias da Próstata/genética , Biópsia , Dano ao DNA , MutagênicosRESUMO
Planar chromatography has recently been combined with six different effect-directed assays for three golden root (Rhodiola rosea L.) samples. However, the profiles obtained showed an intense tailing, making zone differentiation impossible. The profiling was therefore improved to allow for the detection of individual bioactive compounds, and the range of samples was extended to 15 commercial golden root products. Further effect-directed assays were studied providing information on 15 different effect mechanisms, i.e., (1) tyrosinase, (2) acetylcholinesterase, (3) butyrylcholinesterase, (4) ß-glucuronidase, and (5) α-amylase inhibition, as well as endocrine activity via the triplex planar yeast antagonist-verified (6-8) estrogen or (9-11) androgen screen, (12) genotoxicity via the planar SOS-Umu-C bioassay, antimicrobial activity against (13) Gram-negative Aliivibrio fischeri and (14) Gram-positive Bacillus subtilis bacteria, and (15) antioxidative activity (DPPH⢠radical scavengers). Most of the golden root profiles obtained were characteristic, but some samples differed substantially. The United States Pharmacopeia reference product showed medium activity in most of the assays. The six most active compound zones were further characterized using high-resolution mass spectrometry, and the mass signals obtained were tentatively assigned to molecular formulae. In addition to confirming the known activities, this study is the first to report that golden root constituents inhibit butyrylcholinesterase (rosin was tentatively assigned), ß-glucuronidase (rosavin, rosarin, rosiridin, viridoside, and salidroside were tentatively assigned), and α-amylase (stearic acid and palmitic acid were tentatively assigned) and that they are genotoxic (hydroquinone was tentatively assigned) and are both agonistic and antagonistic endocrine active.
Assuntos
Acetilcolinesterase , Butirilcolinesterase , Butirilcolinesterase/farmacologia , Acetilcolinesterase/química , Extratos Vegetais/química , Cromatografia em Camada Fina/métodos , Espectrometria de Massas , Bacillus subtilis , Bioensaio , GlucuronidaseRESUMO
Two herbal plants, Akebia quinata D. leaf/fruit and Clitoria ternatea L. flower, well-known in traditional medicine systems, were investigated using a non-target effect-directed profiling. High-performance thin-layer chromatography (HPTLC) was combined with 11 different effect-directed assays, including two multiplex bioassays, for assessing their bioactivity. Individual active zones were heart-cut eluted for separation via an orthogonal high-performance liquid chromatography column to heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) for tentative assignment of molecular formulas according to literature data. The obtained effect-directed profiles provided information on 2,2-diphenyl-1-picrylhydrazyl scavenging, antibacterial (against Bacillus subtilis and Aliivibrio fischeri), enzyme inhibition (tyrosinase, α-amylase, ß-glucuronidase, butyrylcholinesterase, and acetylcholinesterase), endocrine (agonists and antagonists), and genotoxic (SOS-Umu-C) activities. The main bioactive compound zones in A. quinata leaf were tentatively assigned to be syringin, vanilloloside, salidroside, α-hederin, cuneataside E, botulin, and oleanolic acid, while salidroside and quinatic acids were tentatively identified in the fruit. Taraxerol, kaempherol-3-rutinoside, kaempferol-3-glucoside, quercetin-3-rutinoside, and octadecenoic acid were tentatively found in the C. ternatea flower. This straightforward hyphenated technique made it possible to correlate the biological properties of the herbs with possible compounds. The meaningful bioactivity profiles contribute to a better understanding of the effects and to more efficient food control and food safety.
Assuntos
Clitoria , Acetilcolinesterase/química , Cromatografia em Camada Fina/métodos , Butirilcolinesterase , Extratos Vegetais/química , Espectrometria de Massas por Ionização por Electrospray , BioensaioRESUMO
BACKGROUND & AIMS: Patients with multiple recurrent Clostridioides difficile infection (rCDI) have a disturbed gut microbiota that can be restored by fecal microbiota transplantation (FMT). Despite extensive screening, healthy feces donors may carry bacteria in their intestinal tract that could have long-term health effects, such as potentially procarcinogenic polyketide synthase-positive (pks+) Escherichia coli. Here, we aim to determine whether the pks abundance and persistence of pks+E coli is influenced by pks status of the donor feces. METHODS: In a cohort of 49 patients with rCDI treated with FMT and matching donor samples-the largest cohort of its kind, to our knowledge-we retrospectively screened fecal metagenomes for pks+E coli and compared the presence of pks in patients before and after treatment and to their respective donors. RESULTS: The pks island was more prevalent (P = .026) and abundant (P < .001) in patients with rCDI (pre-FMT, 27 of 49 [55%]; median, 0.46 reads per kilobase per million [RPKM] pks) than in healthy donors (3 of 8 donors [37.5%], 11 of 38 samples [29%]; median, 0.01 RPKM pks). The pks status of patients post-FMT depended on the pks status of the donor suspension with which the patient was treated (P = .046). Particularly, persistence (8 of 9 cases) or clearance (13 of 18) of pks+E coli in pks+ patients was correlated to pks in the donor (P = .004). CONCLUSIONS: We conclude that FMT contributes to pks+E coli persistence or eradication in patients with rCDI but that donor-to-patient transmission of pks+E coli is unlikely.
Assuntos
Clostridioides difficile/patogenicidade , Infecções por Clostridium/terapia , Escherichia coli/crescimento & desenvolvimento , Transplante de Microbiota Fecal , Microbioma Gastrointestinal , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/microbiologia , Disbiose , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transplante de Microbiota Fecal/efeitos adversos , Feminino , Humanos , Masculino , Metagenoma , Metagenômica , Pessoa de Meia-Idade , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Reinfecção , Estudos Retrospectivos , Fatores de Tempo , Resultado do TratamentoRESUMO
The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin produced by pathogenic bacteria causing major foodborne diseases worldwide. CDT activates the DNA Damage Response and modulates the host immune response, but the precise relationship between these outcomes has not been addressed so far. Here, we show that chronic exposure to CDT in HeLa cells or mouse embryonic fibroblasts promotes a strong type I interferon (IFN) response that depends on the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) through the recognition of micronuclei. Indeed, despite active cell cycle checkpoints and in contrast to other DNA damaging agents, cells exposed to CDT reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation in daughter cells. These mitotic phenotypes are observed with CDT from various origins and in cancer or normal cell lines. Finally, we show that CDT exposure in immortalized normal colonic epithelial cells is associated to cGAS protein loss and low type I IFN response, implying that CDT immunomodulatory function may vary depending on tissue and cell type. Thus, our results establish a direct link between CDT-induced DNA damage, genetic instability and the cellular immune response that may be relevant in the context of natural infection associated to chronic inflammation or carcinogenesis.
Assuntos
Toxinas Bacterianas/farmacologia , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Interferon Tipo I/genética , Camundongos , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genéticaRESUMO
Cytolethal distending toxin (CDT) is a bacterial genotoxin that causes host cell cycle arrest and death. We previously employed a Saccharomyces cerevisiae model with inducible expression of the CDT catalytic subunit from Aggregatibacter actinomycetemcomitans, AaCdtB, and showed that a wide variety of host factors play a role in facilitating the activity of CdtB. Our observation that a yeast H2B mutant defective in chromatin condensation was partially resistant to CdtB implies that chromatin structure may affect CDT function. In this study, we identified host chromatin regulatory genes required for CdtB cytotoxicity. We found that the deletion of HTZ1 or certain subunits of SWR, INO80, and SIR complexes increased cellular resistance to CdtB. We hypothesized that CdtB may interact with Htz1 or the chromatin, but immunoprecipitation experiments failed to detect physical interaction between CdtB and Htz1 or the chromatin. However, we observed reduced nuclear localization of CdtB in several mutants, suggesting that impaired nuclear translocation may, at least partly, explain the mechanisms of CdtB resistance. In addition, mutations in chromatin regulatory genes induce changes in the global gene expression profile, and these may indirectly affect CdtB toxicity. Our results suggest that decreased expression of endoplasmic reticulum (ER)-Golgi transport-related genes that may be involved in CdtB transport and/or increased expression of DNA repair genes may contribute to CdtB resistance. These results suggest that the functions of chromatin regulators may contribute to the activity of CDT in host cells.
Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Toxinas Bacterianas/genética , Cromatina/genética , Interações Hospedeiro-Patógeno/genética , Infecções por Pasteurellaceae/genética , Infecções por Pasteurellaceae/microbiologia , Saccharomyces cerevisiae/genética , Toxinas Bacterianas/metabolismo , Cromatina/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mutação , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: Colibactin is a genotoxin that induces DNA double-strand breaks that may lead to carcinogenesis and is produced by Escherichia coli strains harboring the pks island. Human and animal studies have shown that colibactin-producing gut bacteria promote carcinogenesis and enhance the progression of colorectal cancer through cellular senescence and chromosomal abnormalities. In this study, we investigated the impact of prebiotics on the genotoxicity of colibactin-producing E. coli strains Nissle 1917 and NC101. METHODS: Bacteria were grown in medium supplemented with 20, 30 and 40 mg/mL of prebiotics inulin or galacto-oligosaccharide, and with or without 5 µM, 25 µM and 125 µM of ferrous sulfate. Colibactin expression was assessed by luciferase reporter assay for the clbA gene, essential for colibactin production, in E. coli Nissle 1917 and by RT-PCR in E. coli NC101. The human epithelial colorectal adenocarcinoma cell line, Caco-2, was used to assess colibactin-induced megalocytosis by methylene blue binding assay and genotoxicity by γ-H2AX immunofluorescence analysis. RESULTS: Inulin and galacto-oligosaccharide enhanced the expression of clbA in pks+ E. coli. However, the addition of 125 µM of ferrous sulfate inhibited the expression of clbA triggered by oligosaccharides. In the presence of either oligosaccharide, E. coli NC101 increased dysplasia and DNA double-strand breaks in Caco-2 cells compared to untreated cells. CONCLUSION: Our results suggest that, in vitro, prebiotic oligosaccharides exacerbate DNA damage induced by colibactin-producing bacteria. Further studies are necessary to establish whether oligosaccharide supplementation may lead to increased colorectal tumorigenesis in animal models colonized with pks+ E. coli.
Assuntos
Carcinogênese/patologia , Neoplasias do Colo/patologia , Dano ao DNA , Escherichia coli/metabolismo , Mutagênicos/efeitos adversos , Oligossacarídeos/farmacologia , Peptídeos/efeitos adversos , Policetídeos/efeitos adversos , Células CACO-2 , Carcinogênese/induzido quimicamente , Senescência Celular , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Ilhas Genômicas , HumanosRESUMO
Several commensal and pathogenic Gram-negative bacteria produce DNA-damaging toxins that are considered bona fide carcinogenic agents. The microbiota of colorectal cancer (CRC) patients is enriched in genotoxin-producing bacteria, but their role in the pathogenesis of CRC is poorly understood. The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in the majority of sporadic CRCs. We investigated whether the loss of APC alters the response of colonic epithelial cells to infection by Salmonella enterica, the only genotoxin-producing bacterium associated with cancer in humans. Using 2D and organotypic 3D cultures, we found that APC deficiency was associated with sustained activation of the DNA damage response, reduced capacity to repair different types of damage, including DNA breaks and oxidative damage, and failure to induce cell cycle arrest. The reduced DNA repair capacity and inability to activate adequate checkpoint responses was associated with increased genomic instability in APC-deficient cells exposed to the genotoxic bacterium. Inhibition of the checkpoint response was dependent on activation of the phosphatidylinositol 3-kinase pathway. These findings highlight the synergistic effect of the loss of APC and infection with genotoxin-producing bacteria in promoting a microenvironment conducive to malignant transformation.
Assuntos
Polipose Adenomatosa do Colo/genética , Colo/metabolismo , Células Epiteliais/metabolismo , Instabilidade Genômica/genética , Fosfatidilinositol 3-Quinases/metabolismo , Infecções por Salmonella/metabolismo , Salmonella enterica/metabolismo , Polipose Adenomatosa do Colo/microbiologia , Polipose Adenomatosa do Colo/patologia , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Colo/microbiologia , Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/patologia , Dano ao DNA/genética , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Genes Supressores de Tumor/fisiologia , Humanos , Camundongos , Mutagênicos/metabolismo , Infecções por Salmonella/genética , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
Colibactin is a secondary metabolite produced by certain strains of bacteria found in the human gut. The presence of colibactin-producing bacteria has been correlated to colorectal cancer in humans. Colibactin was first discovered in 2006, but because it is produced in small quantities and is unstable, it has yet to be isolated from bacterial cultures. Here we summarize advances in the field since ~2017 that have led to the identification of the structure of colibactin as a heterodimer containing two DNA-reactive electrophilic cyclopropane residues. Colibactin has been shown to form interstrand cross-links by alkylation of adenine residues on opposing strands of DNA. The structure of colibactin contains two thiazole rings separated by a two-carbon linker that is thought to exist as an α-aminoketone following completion of the biosynthetic pathway. However, synthetic studies have now established that this α-aminoketone is unstable toward aerobic oxidation; the resulting oxidation products are in turn unstable toward nucleophilic cleavage under mild conditions. These data provide a simple molecular-level explanation for colibactin's instability and potentially also explain the observation that cell-to-cell contact is required for genotoxic effects.
Assuntos
Proteínas de Escherichia coli , Peptídeos , Policetídeos , Animais , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Estrutura Molecular , Peptídeos/química , Peptídeos/metabolismo , Policetídeos/química , Policetídeos/metabolismoRESUMO
The human gastrointestinal tract is a complex ecosystem in which epithelial cells and microorganisms of the intestinal microbiota live in symbiosis. Certain members of the microbiota, in particular Escherichia coli strains of the B2 phylotype, carry the polyketide synthase-island encoding the genotoxin colibactin. Colibactin is a nonribosomal peptide or polyketide-nonribosomal peptide hybrid of still unsolved structure, which induces DNA double strand breaks (DSBs) in eukaryotic cells. However, direct contact between live bacteria and host cell is required in order to elicit these genotoxic effects. In this study, we used a variety of cell culture models, among them, a 3D cell culture approach based on decellularised small intestinal submucosa, to investigate whether the intestinal mucus layer has the potential to interfere with colibactin activity. We demonstrate that the expression of mucins and the formation of an adherent mucus layer significantly increased with increasing complexity of cell culture. Moreover, we show that the presence of an adherent mucus layer on epithelial cells attenuates the genotoxic activity of colibactin, by preventing the induction of DNA-DSBs. Removal of the adherent mucus layer restored the occurrence of DNA-DSBs.
Assuntos
Trato Gastrointestinal/microbiologia , Muco/microbiologia , Mutagênicos/metabolismo , Peptídeos/metabolismo , Policetídeos/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/fisiologia , Escherichia coli/patogenicidade , Microbioma Gastrointestinal/fisiologia , Ilhas Genômicas/fisiologia , Células HT29 , Humanos , Simbiose/fisiologia , Virulência/fisiologiaRESUMO
A novel protein, soritesidine (SOR) with potent toxicity was isolated from the marine sponge Spongosorites sp. SOR exhibited wide range of toxicities over various organisms and cells including brine shrimp (Artemia salina) larvae, sea hare (Aplysia kurodai) eggs, mice, and cultured mammalian cells. Toxicities of SOR were extraordinary potent. It killed mice at 5 ng/mouse after intracerebroventricular (i.c.v.) injection, and brine shrimp and at 0.34 µg/mL. Cytotoxicity for cultured mammalian cancer cell lines against HeLa and L1210 cells were determined to be 0.062 and 12.11 ng/mL, respectively. The SOR-containing fraction cleaved plasmid DNA in a metal ion dependent manner showing genotoxicity of SOR. Purified SOR exhibited molecular weight of 108.7 kDa in MALDI-TOF MS data and isoelectric point of approximately 4.5. N-terminal amino acid sequence up to the 25th residue was determined by Edman degradation. Internal amino acid sequences for fifteen peptides isolated from the enzyme digest of SOR were also determined. None of those amino acid sequences showed similarity to existing proteins, suggesting that SOR is a new proteinous toxin.
Assuntos
Toxinas Marinhas/toxicidade , Poríferos , Sequência de Aminoácidos , Animais , Aplysia/efeitos dos fármacos , Artemia/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Bioensaio/métodos , Linhagem Celular Tumoral , Humanos , Japão , Larva/efeitos dos fármacos , Masculino , Toxinas Marinhas/administração & dosagem , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Camundongos , Peso Molecular , Testes de Mutagenicidade/métodosRESUMO
Multiple pathogenic Gram-negative bacteria produce the cytolethal distending toxin (CDT) with activity of DNase I; CDT can induce DNA double-strand breaks (DSBs), G2/M cell cycle arrest, and apoptosis in cultured mammalian cells. However, the link of CDT to in vivo tumorigenesis is not fully understood. In this study, 129/SvEv Rag2-/- mice were gavaged with wild-type Helicobacter hepatics 3B1(Hh) and its isogenic cdtB mutant HhcdtBm7, followed by infection for 10 and 20 weeks (WPI). HhCDT deficiency did not affect cecal colonization levels of HhcdtBm7, but attenuated severity of cecal pathology in HhcdtBm7-infected mice. Of importance, preneoplasic dysplasia was progressed to cancer from 10 to 20 WPI in the Hh-infected mice but not in the HhcdtBm7-infected mice. In addition, the loss of HhCDT significantly dampened transcriptional upregulation of cecal Tnfα and Il-6, but elevated Il-10 mRNA levels when compared to Hh at 10 WPI. Furthermore, the presence of HhCDT increased numbers of lower bowel intestinal γH2AX-positive epithelial cells (a marker of DSBs) at both 10 and 20 WPI and augmented phospho-Stat3 foci+ intestinal crypts (activation of Stat3) at 20 WPI. Our findings suggest that CDT promoted Hh carcinogenesis by enhancing DSBs and activation of the Tnfα/Il-6-Stat3 signaling pathway.
Assuntos
Toxinas Bacterianas/metabolismo , Carcinogênese/patologia , Quebras de DNA de Cadeia Dupla , Helicobacter hepaticus/patogenicidade , Interleucina-6/metabolismo , Intestinos/patologia , Fator de Transcrição STAT3/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Toxinas Bacterianas/genética , Ceco/microbiologia , Ceco/patologia , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Helicobacter hepaticus/genética , Histonas/metabolismo , Interleucina-10/genética , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias/microbiologia , Neoplasias/patologia , RNA Mensageiro/biossíntese , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Micronuclei are suitable internal dosimeters for revealing tissue-specific genotoxic damage in individuals exposed to carcinogenic mixtures. Evaluation of radiation-induced cellular changes to predict radiosensitivity has invested many investigators since such changes were first found in biopsy material. AIM: The aim of the present study was to assess the relationship of with histopathological grade and number of radiation therapy sittings with the frequency of micronuclei and nuclear anomalies among oral cancer patients. MATERIAL & METHOD: Thirty male patients with histologically proven cases of oral cancer undergoing radiation therapy and age and sex matched 20 healthy controls were included in the study. Scrape cytology smears of exfoliated buccal cells were prepared and stained using Feulgen stain and frequency of micronuclei and other nuclear anomalies counts were evaluated with the help of light microscope expressed as per 1000 buccal cells. RESULTS: The mean values of the micronuclei and nuclear anomalies were 14.03 and 21.30 respectively. There was a significant association and strong positive correlation of Radiation exposure and grades of squamous cell carcinoma with micronuclei and nuclear anomalies. There was no statistically significant association and correlation between nuclear anomalies in well differentiated and moderately differentiated carcinomas. CONCLUSION: With increase number of radiation therapy sittings, there was increase in number of micronuclei and nuclear anomalies. Hence the result of this study highlights that increased number of micronuclei and nuclear anomalies provides information regarding radiosensitivity of epithelial cells.
Assuntos
Testes para Micronúcleos , Mucosa Bucal/ultraestrutura , Neoplasias Bucais/radioterapia , Bochecha , Humanos , Masculino , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Gradação de TumoresRESUMO
BACKGROUND: Bacterial genotoxins provoke DNA damage and carcinogenesis. The Escherichia coli uropathogenic-specific protein gene, usp, and its linked genes, imu1-3, are associated with strains from pyelonephritis, prostatitis, and bacteremia of urinary tract origin. While the Usp C-terminal domain exhibits similarity with DNase-like colicins and pyocins, its role and mechanisms of action, as well as those of the 3 associated proteins, is unknown. METHODS: We isolated Usp and Imu1-3 and examined their activity on plasmid DNA, human umbilical vein endothelial cells, and human embryonic kidney cells (cell line HEK293). The affect of Usp and Imu1-3 was assessed by MTT and Comet assays, infection assays, caspase 3/7 activity, fluorescently labeled actin staining, and Western blotting. RESULTS: Usp possesses DNase activity and, particularly when coapplied with Imu2, exhibits genotoxic activity in mammalian cells. Infection assays demonstrated that E. coli usp(+) imu1-3(+) affects the viability of mammalian cells, induces increased caspase 3/7 activity, and perturbs cell cytoskeleton structure. CONCLUSIONS: Usp is a novel E. coli genotoxin active against mammalian cells. Optimal in vivo activity of Usp requires Imu2. Infection with E. coli usp(+) imu1-3(+) induces a response characteristic of apoptosis.
Assuntos
Bacteriocinas/farmacologia , Proteínas de Escherichia coli/farmacologia , Mutagênicos/farmacologia , Bacteriocinas/toxicidade , Caspases/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Desoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Mutagênicos/toxicidadeRESUMO
Drosophila melanogaster is one of the crucial in vivo models in terms of analyzing the toxicity of various unknown chemicals. Every part of the fly serves as a model in metabolic and therapeutic approaches. Genotoxic and teratogenic compounds are exposed to Drosophila through the oral route. Further, the toxicity of genotoxic compounds is analyzed in Drosophila's gut, hemolymph, and phenotype. The toxicity of teratogen compounds is also analyzed using a Drosophila embryo. The current chapter summarizes several techniques that are used to detect the genotoxicity and teratogenicity of any unknown compound in this model.
Assuntos
Teratogênese , Teratogênicos , Animais , Teratogênicos/toxicidade , Drosophila melanogaster/genética , Drosophila , Dano ao DNARESUMO
Disinfection by-products (DBPs) are a concern due to their presence in chlorinated wastewater, sewage treatment plant discharge, and surface water, and their potential for environmental toxicity. Despite some attention to their ecotoxicity, little is known about the phytotoxicity of DBPs. This study aimed to evaluate the individual and combined phytotoxicity of four trihalomethanes (THMs: trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM) and their mixture (THM4)), and trichloroacetic acid (TCAA) using genotoxic and cytotoxic assays. The analysis included seed germination tests using Vigna radiata and root growth tests, mitosis studies, oxidative stress response, chromosomal aberrations (CA), and DNA laddering using Allium cepa. The results showed a progressive increase in root growth inhibition for both plant species as the concentration of DBPs increased. High concentrations of mixtures of four THMs resulted in significant (p < 0.05) antagonistic interactions. The effective concentration (EC50) value for V. radiata was 5655, 3145, 2690, 1465, 3570, and 725 mg/L for TCM, BDCM, DBCM, TBM, THM4, and TCAA, respectively. For A. cepa, the EC50 for the same contaminants was 700, 400, 350, 250, 450, and 105 mg/L, respectively. DBP cytotoxicity was observed through CAs, including C-metaphase, unseparated anaphase, lagging chromosome, sticky metaphase, and bridging. Mitotic depression (MD) increased with dose, reaching up to 54.4% for TCAA (50-500 mg/L). The electrophoresis assay showed DNA fragmentation and shearing, suggesting genotoxicity for some DBPs. The order of phytotoxicity for the tested DBPs was TCAA > TBM > DBCM > BDCM > THM4 > TCM. These findings underscore the need for further research on the phytotoxicity of DBPs, especially given their common use in agricultural practices such as irrigation and the use of sludge as manure.
Assuntos
Vigna , Poluentes Químicos da Água , Ácido Tricloroacético/toxicidade , Cebolas , Trialometanos/toxicidade , Desinfecção/métodos , Clorofórmio , Poluentes Químicos da Água/toxicidadeRESUMO
Emerging evidence indicates bacterial infections contribute to the formation of cancers. Bacterial genotoxins are effectors that cause DNA damage by introducing single- and double-strand DNA breaks in the host cells. The first bacterial genotoxin cytolethal distending toxin (CDT) was a protein identified in 1987 in a pathogenic strain in Escherichia coli (E. coli) isolated from a young patient. The peptide-polyketide genotoxin colibactin is produced by the phylogenetic group B2 of E. coli. Recently, a protein produced by attaching/effacing (A/E) pathogens, including enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC) and their murine equivalent Citrobacter rodentium (CR), has been reported as a novel protein genotoxin, being injected via the type III secretion system (T3SS) into host cells and harboring direct DNA digestion activity with a catalytic histidine-aspartic acid dyad. These E. coli-produced genotoxins impair host DNA, which results in senescence or apoptosis of the target cells if the damage is beyond repair. Conversely, host cells can survive and proliferate if the genotoxin-induced DNA damage is not severe enough to kill them. The surviving cells may accumulate genomic instability and acquire malignant traits. This review presents the cellular responses of infection with the genotoxins-producing E. coli and discusses the current knowledge of the tumorigenic potential of these toxins.
RESUMO
The etiology of colorectal cancer (CRC) is influenced by bacterial communities that colonize the gastrointestinal tract. These microorganisms derive essential nutrients from indigestible dietary or host-derived compounds and activate molecular signaling pathways necessary for normal tissue and immune function. Associative and mechanistic studies have identified bacterial species whose presence may increase CRC risk, including notable examples such as Fusobacterium nucleatum, Enterotoxigenic Bacteroides fragilis, and pks+ E. coli. In recent years this work has expanded in scope to include aspects of host mutational status, intra-tumoral microbial heterogeneity, transient infection, and the cumulative influence of multiple carcinogenic bacteria after sequential or co-colonization. In this review, we will provide an updated overview of how host-bacteria interactions influence CRC development, how this knowledge may be utilized to diagnose or prevent CRC, and how the gut microbiome influences CRC treatment efficacy.