Genotoxic Molecules Research Articles

Metabolomics study of the formation of genotoxic molecules based on the fecal volatile metabolites profile using an in vivo animal model

Epidemiological studies have shown that haem iron from processed meat is a key element involved in the colon carcinogenesis. The haem iron induces lipid peroxidation in the colon lumen during digestion, enabling the formation of cytotoxic molecules derived from these reactions. The cytotoxic molecules generated are highly dependent on dietary factors such as lipid sources, calcium levels or the presence of antioxidants during digestion. Here, we investigated whether nitrite substitution by polyphenols as a food additive could modulate the in vivo luminal lipid peroxidation in the colon and consequently, reduce the formation of mucin-depleted foci in a chemical-induced colon cancer rat model. The addition of polyphenols to the cooked ham diet reduces the lipid peroxidation in the rats. A reduction in cytotoxic aldehydes in fecal water from animals fed with polyphenols as well as a decrease in the formation of mucin-depleted foci is observed. The antioxidant capacity derived from polyphenols modifies the luminal environment of the colon, allowing the identification of a specific molecular signature derived from the analysis of fecal volatile organic compounds. In this molecular signature is included the reduction in the abundance of (2E,4E)-2,4-hexadienal, a carcinogenic aldehyde derived from lipid peroxidation.

Azadirachta indica A. Juss (neem) phenolic extract inhibits human B-lymphoblastoid cells growth via cell cycle arrest, apoptosis induction, and DNA damage

Aim: As far as is known, the pharmaceutical effects of neem on human B-lymphoblastoid (TK6) cells have not been studied until now. Hence, the present study aimed to obtain neem phenolic extracts for inhibits the proliferation of TK6 cells and explore some possible underlying mechanisms involved in these effects. Methods: Hexane extract (HE) was obtained in the first step. After that, the residual hexane was removed from the neem. The dried neem sample was used in a new extraction for obtaining the ethyl acetate extract (EAE). Total phenolic compounds (TPC) and total flavonoid contents (TFC) were determined by spectrophotometric methods. Lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests were used to evaluate the cytotoxicity in TK6 cells. The stop at G0/G1 cell cycle and inducing apoptosis in the TK6 cells were analyzed by flow cytometry. For deoxyribonucleic acid (DNA) damage evaluation, the alkaline comet test was used. Results: The higher TFC (65.50 mg/g of extract ± 1.17 mg/g of extract) and TPC (52.08 mg of extract ± 0.88 mg of extract) were obtained in EAE compared to HE that was obtained TFC of 14.61 mg/g of extract ± 0.60 mg/g of extract and TPC of 3.20 mg/g of extract ± 1.20 mg/g of extract. EAE was more significantly cytotoxic to TK6 cells than HE. The apoptosis induction was higher after exposure to 15.0 µg/mL of EAE (11.29%) in comparison to 15.0 µg/mL of HE (2.52%). The G0/G1 phase increased from 72% negative control (NC) to 83% after treatment with neem extracts (15 µg/mL). Neem extracts were also able to cause DNA strand breaks in TK6 cells. Conclusions: The extraction residue from neem leaf after hexane extraction is a source important of cytotoxic and genotoxic molecules against TK6 cells, the results also can suggest that the toxic effects in TK6 cells can be provided most likely due to the presence of high content of TPC from neem extracts.

Commensal microbiota from patients with inflammatory bowel disease produce genotoxic metabolites.

Microbiota-derived metabolites that elicit DNA damage can contribute to colorectal cancer (CRC). However, the full spectrum of genotoxic chemicals produced by indigenous gut microbes remains to be defined. We established a pipeline to systematically evaluate the genotoxicity of an extensive collection of gut commensals from inflammatory bowel disease patients. We identified isolates from divergent phylogenies whose metabolites caused DNA damage and discovered a distinctive family of genotoxins-termed the indolimines-produced by the CRC-associated species Morganella morganii. A non-indolimine-producing M. morganii mutant lacked genotoxicity and failed to exacerbate colon tumorigenesis in mice. These studies reveal the existence of a previously unexplored universe of genotoxic small molecules from the microbiome that may affect host biology in homeostasis and disease.

Escherichia coli Strains from Patients with Inflammatory Bowel Diseases have Disease-specific Genomic Adaptations.

Escherichia coli is over-abundant in the gut microbiome of patients with inflammatory bowel disease [IBD]. Here, we aimed to identify IBD-specific genomic functions of diverse E. coli lineages. We investigated E. coli genomes from patients with ulcerative colitis [UC], Crohn's disease [CD] or a pouch, and healthy subjects. The majority of genomes were reconstructed from metagenomic samples, including newly sequenced faecal metagenomes. Clinical metadata were collected. Functional analysis at the gene and mutation level were performed and integrated with IBD phenotypes and biomarkers. Overall, 530 E. coli genomes were analysed. The E. coli B2 lineage was more prevalent in UC compared with other IBD phenotypes. Genomic metabolic capacities varied across E. coli lineages and IBD phenotypes. Host mucin utilisation enzymes were present in a single lineage and depleted in patients with a pouch, whereas those involved in inulin hydrolysis were enriched in patients with a pouch. E. coli strains from patients with UC were twice as likely to encode the genotoxic molecule colibactin than strains from patients with CD or a pouch. Strikingly, patients with a pouch showed the highest inferred E. coli growth rates, even in the presence of antibiotics. Faecal calprotectin did not correlate with the relative abundance of E. coli. Finally, we identified multiple IBD-specific non-synonymous mutations in E. coli genes encoding for bacterial cell envelope components. Comparative genomics indicates that E. coli is a commensal species adapted to the overactive mucosal immune milieu in IBD, rather than causing it. Our results reveal mutations that may lead to attenuated antigenicity in some E. coli strains.

Tucumã (Astrocaryum aculeatum) Prevents Oxidative and DNA Damage to Retinal Pigment Epithelium Cells.

Eye diseases have a negative impact on the eyesight quality of the world population. The age-related macular degeneration (AMD) draws special attention since it is a chronic disorder characterized by oxidative and inflammatory damage to the retinal epithelial pigment, which triggers progressive vision loss. In the Brazilian Amazon, Astrocaryum aculeatum is an Amazonian fruit (Tucumã) used by riverside communities in traditional medicine to treat a number of ailments. These communities have recently shown to have increased longevity and reduced prevalence of age-related morbidity. Thus, the aim of this research was to chemically characterize and analyze the in vitro antioxidant effect and molecular damage prevention of the Tucumã ethanolic extract in retinal pigment epithelium (RPE) cells in a model for AMD. The extract was chemically characterized by ultra-high-performance liquid chromatography (HPLC) coupled with diode-array detection and mass spectrophotometry (HPLC-DAD-MS). In vitro protocols were performed, and the cytopreventive effect of Tucumã on RPE cells exposed to high concentrations of superoxide anion, an oxidant and genotoxic molecule, as well as the effect of Tucumã extract on oxidative and molecular makers were assessed. Biochemical and flow cytometry analyses were conducted in these protocols. The extract presents high concentrations of caffeic acid, gallic acid, catechin, luteolin, quercetin, and rutin. Treatment did not show cytotoxic effects in cells treated only with extract at 50 μg/mL. In fact, it improved cell viability and was able to prevent necrosis and apoptosis, and oxidative and molecular damage was significantly reduced. In summary, Tucumã is an important Amazon fruit, which seems to contribute significantly to improve human health conditions, as our findings suggest that its extract has a relevant chemical matrix rich in antioxidant molecules, and its consumption could improve eye health and contribute to prevention against oxidative stress through cytoprevention, reactive oxygen species reduction, and maintenance of DNA integrity in retinal pigment epithelium (RPE) cells.

In Vitro Biological Properties of Solanum sessiliflorum (Dunal), an Amazonian Fruit.

Solanum sessiliflorum is an Amazonian fruit (cubiu) that has been domesticated since pre-Colombian era. It is also used in folk medicine to treat some clinical conditions. This investigation chemically characterized and analyzed the in vitro antioxidant and antitumoral effect of a cubiu pulp/seed hydroalcoholic extract. Cubiu extract was chemically characterized by high-performance liquid chromatography with diode array detector (HPLC-DAD), its antioxidant capacity measured by 2.2-diphenyl-1-picrylhydrazyl (DPPH) assay, and the following complementary in vitro protocols were performed: (1) cytoprotective effect of cubiu on human peripheral blood mononuclear cells (PBMCs) exposed to H2O2, a genotoxic and procarcinogen molecule; (2) effect of cubiu on low density lipoproteins oxidation; and (3) cytotoxic and antiproliferative effect on breast (MCF-7) and colorectal (HT-29) cancer cell lines. Biochemical and flow cytometry analyses were conducted in these protocols. Cubiu extract presented high concentrations of caffeic and gallic acids, beta-carotene, catechin, quercetin, and rutin, and its antioxidant capacity was confirmed. Cubiu attenuated H2O2 cytotoxicity on PBMCs, presented lowering effect on LDL oxidation, and induced mortality and proliferative inhibition of colorectal cancer cells. In cancer cells, cubiu extract at 10 μg/mL showed similar effects to 5-fluorouracil chemo drug reducing its viability and frequency of S-phase, indicating that cells are undergoing mitosis. In summary, despite the limitations of in vitro protocols, our results suggest that cubiu has several biological properties that affect human health.

Carriage of Colibactin-producing Bacteria and Colorectal Cancer Risk.

Colibactin is a genotoxic molecule, produced primarily by Escherichia coli. Colibactin causes DNA damage that may lead to colorectal cancer. Here we review recent advances in the study of colibactin and propose a focus on patients with inflammatory bowel diseases (IBD) who have higher levels of colibactin-producing bacteria in their intestines.

Genotoxic stress increases cytoplasmic mitochondrial DNA editing by human APOBEC3 mutator enzymes at a single cell level

Human cells are stressed by numerous mechanisms that can lead to leakage of mitochondrial DNA (mtDNA) to the cytoplasm and ultimately apoptosis. This agonist DNA constitutes a danger to the cell and is counteracted by cytoplasmic DNases and APOBEC3 cytidine deamination of DNA. To investigate APOBEC3 editing of leaked mtDNA to the cytoplasm, we performed a PCR analysis of APOBEC3 edited cytoplasmic mtDNA (cymtDNA) at the single cell level for primary CD4+ T cells and the established P2 EBV blast cell line. Up to 17% of primary CD4+ T cells showed signs of APOBEC3 edited cymtDNA with ~50% of all mtDNA sequences showing signs of APOBEC3 editing – between 1500–5000 molecules. Although the P2 cell line showed a much lower frequency of stressed cells, the number of edited mtDNA molecules in such cells was of the same order. Addition of the genotoxic molecules, etoposide or actinomycin D increased the number of cells showing APOBEC3 edited cymtDNA to around 40%. These findings reveal a very dynamic image of the mitochondrial network, which changes considerably under stress. APOBEC3 deaminases are involved in the catabolism of mitochondrial DNA to circumvent chronic immune stimulation triggered by released mitochondrial DNA from damaged cells.

Abstract 1405: A novel chemotherapeutic agent to treat tumors with DNA mismatch repair deficiencies

Abstract Impairing the division of cancer cells with genotoxic small molecules has been a primary goal to develop chemotherapeutic agents. However, DNA mismatch repair (MMR)-deficient cancer cells, are resistant to most conventional chemotherapeutic agents. Here we have identified baicalein as a small molecule that selectively kills MutSα-deficient cancer cells. Baicalein binds preferentially to mismatched DNA and induces a DNA damage response in a mismatch repair-dependent manner. In MutSα-proficient cells, baicalein binds to MutSα to dissociate CHK2 from MutSα leading to S phase arrest and cell survival. In contrast, continued replication in the presence of baicalein in MutSα-deficient cells results in a high number of DNA double-strand breaks and ultimately leads to apoptosis. Consistently, baicalein specifically shrinks MutSα-deficient xenograft tumors and inhibits the growth of AOM-DSS-induced colon tumors in colon-specific MSH2 knockout mice. Collectively, baicalein offers the potential of an improved treatment option for patients with tumors with a DNA MMR deficiency. Citation Format: Kyungjae Myung, Yongliang Zhang, Young-Un Park. A novel chemotherapeutic agent to treat tumors with DNA mismatch repair deficiencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1405. doi:10.1158/1538-7445.AM2017-1405

Colibactin Contributes to the Hypervirulence of pks+ K1 CC23 Klebsiella pneumoniae in Mouse Meningitis Infections.

Klebsiella pneumoniae is the most common pathogen of community-acquired meningitis in Taiwan. However, the lack of a physiologically relevant meningitis model for K. pneumoniae has impeded research into its pathogenesis mechanism. Based on the core genome MLST analyses, the hypervirulent K1 K. pneumoniae strains, which are etiologically implicated in adult meningitis, mostly belong to a single clonal complex, CC23. Some K1 CC23 K. pneumoniae strains carry a gene cluster responsible for colibactin production. Colibactin is a small genotoxic molecule biosynthesized by an NRPS-PKS complex, which is encoded by genes located on the pks island. Compared to other hypervirulent K. pneumoniae which primarily infect the liver, the colibactin-producing (pks+) K1 CC23 strains had significant tropism toward the brain of BALB/c mice. We aimed in this study to develop a physiologically relevant meningitis model with the use of pks+ K1 CC23 K. pneumoniae. Acute meningitis was successfully induced in adult BALB/c male mice through orogastric, intranasal, and intravenous inoculation of pks+ K1 CC23 K. pneumoniae. Besides the typical symptoms of bacterial meningitis, severe DNA damages, and caspase 3-independent cell death were elicited by the colibactin-producing K1 CC23 K. pneumoniae strain. The deletion of clbA, which abolished the production of colibactin, substantially hindered K. pneumoniae hypervirulence in the key pathogenic steps toward the development of meningitis. Our findings collectively demonstrated that colibactin was necessary but not sufficient for the meningeal tropism of pks+ K1 CC23 K. pneumoniae, and the mouse model established in this study can be applied to identify other virulence factors participating in the development of this life-threatening disease.

ClbM is a versatile, cation-promiscuous MATE transporter found in the colibactin biosynthetic gene cluster

Multidrug transporters play key roles in cellular drug resistance to toxic molecules, yet these transporters are also involved in natural product transport as part of biosynthetic clusters in bacteria and fungi. The genotoxic molecule colibactin is produced by strains of virulent and pathobiont Escherichia coli and Klebsiella pneumoniae. In the biosynthetic cluster is a multidrug and toxic compound extrusion protein (MATE) proposed to transport the prodrug molecule precolibactin across the cytoplasmic membrane, for subsequent cleavage by the peptidase ClbP and cellular export. We recently determined the X-ray structure of ClbM, and showed preliminary data suggesting its specific role in precolibactin transport. Here, we define a functional role of ClbM by examining transport capabilities under various biochemical conditions. Our data indicate ClbM responds to sodium, potassium, and rubidium ion gradients, while also having substantial transport activity in the absence of alkali cations.

A Novel Chemotherapeutic Agent to Treat Tumors with DNA Mismatch Repair Deficiencies.

Impairing the division of cancer cells with genotoxic small molecules has been a primary goal to develop chemotherapeutic agents. However, DNA mismatch repair (MMR)-deficient cancer cells are resistant to most conventional chemotherapeutic agents. Here we have identified baicalein as a small molecule that selectively kills MutSα-deficient cancer cells. Baicalein binds preferentially to mismatched DNA and induces a DNA damage response in a MMR-dependent manner. In MutSα-proficient cells, baicalein binds to MutSα to dissociate CHK2 from MutSα leading to S-phase arrest and cell survival. In contrast, continued replication in the presence of baicalein in MutSα-deficient cells results in a high number of DNA double-strand breaks and ultimately leads to apoptosis. Consistently, baicalein specifically shrinks MutSα-deficient xenograft tumors and inhibits the growth of AOM-DSS-induced colon tumors in colon-specific MSH2 knockout mice. Collectively, baicalein offers the potential of an improved treatment option for patients with tumors with a DNA MMR deficiency. Cancer Res; 76(14); 4183-91. ©2016 AACR.

Aldehydes with high and low toxicities inactivate cells by damaging distinct cellular targets

Aldehydes are genotoxic and cytotoxic molecules and have received considerable attention for their associations with the pathogenesis of various human diseases. In addition, exposure to anthropogenic aldehydes increases human health risks. The general mechanism of aldehyde toxicity involves adduct formation with biomolecules such as DNA and proteins. Although the genotoxic effects of aldehydes such as mutations and chromosomal aberrations are directly related to DNA damage, the role of DNA damage in the cytotoxic effects of aldehydes is poorly understood because concurrent protein damage by aldehydes has similar effects. In this study, we have analysed how saturated and α,β-unsaturated aldehydes exert cytotoxic effects through DNA and protein damage. Interestingly, DNA repair is essential for alleviating the cytotoxic effect of weakly toxic aldehydes such as saturated aldehydes but not highly toxic aldehydes such as long α,β-unsaturated aldehydes. Thus, highly toxic aldehydes inactivate cells exclusively by protein damage. Our data suggest that DNA interstrand crosslinks, but not DNA-protein crosslinks and DNA double-strand breaks, are the critical cytotoxic DNA damage induced by aldehydes. Further, we show that the depletion of intracellular glutathione and the oxidation of thioredoxin 1 partially account for the DNA damage-independent cytotoxicity of aldehydes. On the basis of these findings, we have proposed a mechanistic model of aldehyde cytotoxicity mediated by DNA and protein damage.

MATE transport of the E. coli-derived genotoxin colibactin.

Various forms of cancer have been linked to the carcinogenic activities of microorganisms(1-3). The virulent gene island polyketide synthase (pks) produces the secondary metabolite colibactin, a genotoxic molecule(s) causing double-stranded DNA breaks(4) and enhanced colorectal cancer development(5,6). Colibactin biosynthesis involves a prodrug resistance strategy where an N-terminal prodrug scaffold (precolibactin) is assembled, transported into the periplasm and cleaved to release the mature product(7-10). Here, we show that ClbM, a multidrug and toxic compound extrusion (MATE) transporter, is a key component involved in colibactin activity and transport. Disruption of clbM attenuated pks+ E. coli-induced DNA damage in vitro and significantly decreased the DNA damage response in gnotobiotic Il10(-/-) mice. Colonization experiments performed in mice or zebrafish animal models indicate that clbM is not implicated in E. coli niche establishment. The X-ray structure of ClbM shows a structural motif common to the recently described MATE family. The 12-transmembrane ClbM is characterized as a cation-coupled antiporter, and residues important to the cation-binding site are identified. Our data identify ClbM as a precolibactin transporter and provide the first structure of a MATE transporter with a defined and specific biological function.

Anti-genotoxic ability of α-tocopherol and Anthocyanin to counteract fish DNA damage induced by musk xylene.

Many compounds released into the environment are able to interact with genetic material. The main purpose of genetic toxicology is to investigate the adverse effects of genotoxic molecules such as reduced fitness, changes in gene frequencies and their impact on genetic diversity in populations following genotoxic exposure. However, the ecological effects of many genotoxic compounds remain poorly understood. The aim of this research was to evaluate the genotoxic activity of an artificial musk (musk xylene, MX) and the potential anti-genotoxicity against this chemical compound of two antioxidant substances (α-tocopherol and an anthocyanins enriched extract). The studies were performed both in vivo and in vitro, using the teleost Danio rerio and the DLEC (Dicentrarchus labrax embryonic cells) cell line. We carried out the exposure to these substances at different times. DNA and cell damage and their possible repair were detected by various experimental approaches: DNA strand breaks (Comet Assay), degree of apoptosis (Diffusion Assay) and molecular alterations at the genomic level (RAPD-PCR technique). Data were collected and analyzed for statistical significance using the Student's t test. The results of this study showed that MX exhibited a genotoxic activity even after short exposure times. The anti-genotoxicity experiments evidenced that both α-tocopherol and Anthocyanin were able to contrast the genotoxic effects induced by MX, both in vivo and in vitro.

Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening.

In a previous study, we validated an in vitro genotoxicity assay based on γH2AX quantification using the In-Cell Western (ICW) method in HepG2 cells. The assay demonstrated high sensitivity and specificity but failed to detect genotoxicity for few compounds that require specific metabolic bioactivation not sufficiently covered by HepG2 cells. The aim of the present study was to assess γH2AX ICW sensitivity using a broader range of genotoxic molecules with HepG2 cells and three additional human cell lines with distinct biotransformation properties: two cell lines expressing some phase I and II bioactivation capabilities (LS-174T and Hep3B), and one with poor general bioactivation properties (ACHN). We evaluated the four cell lines by testing 24 compounds recommended by European Centre for the Validation of Alternative Methods and a set of 24 additional chemicals with different mode of genotoxic action (MOA) (aneugenicity, DNA adducts formation, induction of oxidative stress), including some known to require specific cytochrome P450 metabolic bioactivation. Results for the 48 compounds tested showed that the γH2AX ICW assay was more sensitive with LS-174T and HepG2 cells than with Hep3B or ACHN cell lines. Among the 38 compounds tested with positive or equivocal carcinogenicity data, 36 (95%) showed a positive genotoxic response with the γH2AX ICW assay compared to only 27 (71%) using the Ames assay. We confirm that the γH2AX ICW assay on HepG2 cells, without an exogenous metabolic activation system, may be a suitable test to predict the in vivo genotoxicity of chemicals with different genotoxic MOA. Moreover, the use of the ACHN cell line in combination with LS-174T and HepG2 cells may permit in many cases to discriminate direct from bioactivated genotoxins. Overall, our results confirm the high sensitivity of the γH2AX ICW assay which, in turn, should reduce the number of animals used for genotoxicity assessment.

In Vitro Inhibition of 4-Nitroquinoline-1-Oxide Genotoxicity by Probiotic Lactobacillus rhamnosus IMC501.

Inhibition of 4-nitroquinoline-1-oxide (4-NQO) genotoxicity by a probiotic strain of Lactobacillus rhamnosus (IMC501) was assessed by the prokaryotic short-term bioassay SOSChromotest, using Escherichia coli PQ37 as the target organism. Results showed the ability of strain IMC501 to rapidly and markedly counteract, in vitro, the DNA damage originated by the considered genotoxin. The inhibition was associated with a spectroscopic hypsochromic shift of the original 4-NQO profile and progressive absorbance increase of a new peak. IR-Raman and GC-MS analyses confirmed the disappearance of 4-NQO after contact with the microorganism, showing also the absence of any genotoxic molecule potentially available for metabolic activation (i.e., 4-hydroxyaminoquinoline-1-oxide and 4-nitrosoquinoline-1-oxide). Furthermore, we have shown the presence of the phenyl-quinoline and its isomers as major non-genotoxic conversion products, which led to the hypothesis of a possible pattern of molecular transformation. These findings increase knowledge on lactobacilli physiology and contribute to the further consideration of antigenotoxicity as a nonconventional functional property of particular probiotic strains.

The fungal secondary metabolite alternariol and Alternaria induced immunity (MPF6P.659)

Abstract Sensitivity to the airborne fungus Alternaria alternata (common mold) is believed to be a common cause of allergic asthma. Epidemiological studies worldwide indicate that Alternaria sensitivity is closely linked with the development and exacerbation of allergic airway disorders such as asthma, allergic rhinitis, and chronic rhinosinusitis. Therapies are limited by the lack of knowledge about the role of individual fungal gene products in airway responses. The A. alternata mycotoxin alternariol is a genotoxic and cytotoxic molecule and a major contaminant of most food, grains and feed products. We hereby present a study where we explored the hypothesis of alternariol having immune-modulatory properties. It led us to the discovery that alternariol is strongly anti-inflammatory. We have investigated alternariol’s dynamic interplay between innate immunity and human bronchoalveolar epithelial cells. Immune assays have provided the key doses needed to suppress LPS-induced inflammation. We have also profiled the response of mutant fungal spores that no longer produce alternariol with normal wildtype spores on lung epithelium. This model has also been used to investigate alternariol’s mechanism of action in relation to aryl hydrocarbon receptor. Greater understanding of the role of alternariol in allergic asthma may lead to improved treatment strategies as well as a potential drug target for inflammatory syndromes.

The colibactin warhead crosslinks DNA.

Members of the human microbiota are increasingly being correlated to human health and disease states, but the majority of the underlying microbial metabolites that regulate host-microbe interactions remain largely unexplored. Select strains of E. coli present in the human colon have been linked to initiating inflammation-induced colorectal cancer through an unknown small molecule-mediated process. The responsible nonribosomal peptide-polyketide hybrid pathway encodes “colibactin,” a largely uncharacterized family of small molecules. Genotoxic small molecules from this pathway capable of initiating cancer formation have remained elusive due to their high instability. Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labeling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure. The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin’s DNA-damaging activity. The data support unexpected models for both colibactin biosynthesis and its mode of action.

Non-Genotoxic Assessment of a Natural Antimicrobial Agent: Squalamine

Squalamine, a natural aminosterol compound isolated from the dogfish shark Squalus acanthias, is reported to possess high antimicrobial activities. Herein, we assessed its in vitro genotoxic properties by using the Ames test and the micronucleus assay. All the experimental results demonstrate that squalamine is a non genotoxic molecule and call for further work to evaluate its potent human therapeutic use as a new class of antinfectious agents. Keywords: Ames test, antimicrobial agent, non-genotoxic, squalamine.