The hCOMET project: International database comparison of results with the comet assay in human biomonitoring. Baseline frequency of DNA damage and effect of main confounders.

The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations.

Autophagy: A Player in response to Oxidative Stress and DNA Damage.

Autophagy is a catabolic pathway activated in response to different cellular stressors, such as damaged organelles, accumulation of misfolded or unfolded proteins, ER stress, accumulation of reactive oxygen species, and DNA damage. Some DNA damage sensors like FOXO3a, ATM, ATR, and p53 are known to be important autophagy regulators, and autophagy seems therefore to have a role in DNA damage response (DDR). Recent studies have partly clarified the pathways that induce autophagy during DDR, but its precise role is still not well known. Previous studies have shown that autophagy alterations induce an increase in DNA damage and in the occurrence of tumor and neurodegenerative diseases, highlighting its fundamental role in the maintenance of genomic stability. During DDR, autophagy could act as a source of energy to maintain cell cycle arrest and to sustain DNA repair activities. In addition, autophagy seems to play a role in the degradation of components involved in the repair machinery. In this paper, molecules which are able to induce oxidative stress and/or DNA damage have been selected and their toxic and genotoxic effects on the U937 cell line have been assessed in the presence of the single compounds and in concurrence with an inhibitor (chloroquine) or an inducer (rapamycin) of autophagy. Our data seem to corroborate the fundamental role of this pathway in response to direct and indirect DNA-damaging agents. The inhibition of autophagy through chloroquine had no effect on the genotoxicity induced by the tested compounds, but it led to a high increase of cytotoxicity. The induction of autophagy, through cotreatment with rapamycin, reduced the genotoxic activity of the compounds. The present study confirms the cytoprotective role of autophagy during DDR; its inhibition can sensitize cancer cells to DNA-damaging agents. The modulation of this pathway could therefore be an innovative approach able to reduce the toxicity of many compounds and to enhance the activity of others, including anticancer drugs.

Is the protein profile of pig Longissimus dorsi affected by gender and diet?

The impact of gender and diet on the proteome of Longissimus dorsi was addressed by 2D-PAGE analysis of male and female pigs, fed with a barley-based control diet and a diet enriched with extruded linseed and plant extracts. No statistically significant difference in protein number between female and male samples was found. Furthermore, PCA excluded gender-dependent protein clusters. For both the control and enriched diet, several spots exhibited at least a 1.5-fold intensity difference, but none showed a statistically relevant variation. Protein profiles PCA for both diets indicated that the first two principal components account up to 47% of total variance, with two diet-dependent separated clusters. Among 176 common spots, 29 exhibited >1.5 fold change, mostly more abundant in the control diet. PMF identified 14 distinct proteins, including myofibrillar proteins, glycolytic enzymes and myoglobin, thus suggesting a diet-dependent meat quality. A statistically significant increase in carbonylated proteins of enriched diet samples was detected using the 2,4-dinitrophenylhydrazine method but not using fluorescein-5-thiosemicarbazide-labeled bands. ROS induction and DNA oxidative damage, detected in a human cell line exposed to digested meat from both diets, further support the notion that the enriched diet does not protect against oxidative stress. SIGNIFICANCE: The comparison of the protein profile of female and male Longissimus dorsi from pigs fed by a control diet and a diet enriched with polyphenols, indicate no gender effect, whereas diet affects the abundance of several proteins, possibly linked to meat quality. Protein carbonylation was statistically higher in meat from the enriched diet, suggesting that polyphenols at the concentration present in the diet did not exert a protective effect against oxidation.

The Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid (SAHA) Restores Cardiomyocyte Contractility in a Rat Model of Early Diabetes.

In early diabetes, hyperglycemia and the associated metabolic dysregulation promote early changes in the functional properties of cardiomyocytes, progressively leading to the appearance of the diabetic cardiomyopathy phenotype. Recently, the interplay between histone acetyltransferases (HAT) and histone deacetylases (HDAC) has emerged as a crucial factor in the development of cardiac disorders. The present study evaluates whether HDAC inhibition can prevent the development of cardiomyocyte contractile dysfunction induced by a short period of hyperglycemia, with focus on the potential underlying mechanisms. Cell contractility and calcium dynamics were measured in unloaded ventricular myocytes isolated from the heart of control and diabetic rats. Cardiomyocytes were either untreated or exposed to the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) for 90 min. Then, a fraction of each group of cells was used to evaluate the expression levels of proteins involved in the excitation-contraction coupling, and the cardiomyocyte metabolic activity, ATP content, and reactive oxygen species levels. SAHA treatment was able to counteract the initial functional derangement in cardiomyocytes by reducing cell oxidative damage. These findings suggest that early HDAC inhibition could be a promising adjuvant approach for preventing diabetes-induced cardiomyocyte oxidative damage, which triggers the pro-inflammatory signal cascade, mitochondrial damage, and ventricular dysfunction.

Engineering methionine γ-lyase from Citrobacter freundii for anticancer activity.

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in KM, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC50 lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis.

Imatinib mesylate-induced cardiomyopathy involves resident cardiac progenitors.

Cardiovascular complications are included among the systemic effects of tyrosine kinase inhibitor (TKI)-based therapeutic strategies. To test the hypothesis that inhibition of Kit tyrosine kinase that promotes cardiac progenitor cell (CPC) survival and function may be one of the triggering mechanisms of imatinib mesylate (IM)-related cardiovascular effects, the anatomical, structural and ultrastructural changes in the heart of IM-treated rats were evaluated. Cardiac anatomy in IM-exposed rats showed a dose-dependent, restrictive type of remodeling and depressed hemodynamic performance in the absence of remarkable myocardial fibrosis. The effects of IM on rat and human CPCs were also assessed. IM induced rat CPC depletion, reduced growth and increased cell death. Similar effects were observed in CPCs isolated from human hearts. These results extend the notion that cardiovascular side effects are driven by multiple actions of IM. The identification of cellular mechanisms responsible for cardiovascular complications due to TKIs will enable future strategies aimed at preserving concomitantly cardiac integrity and anti-tumor activity of advanced cancer treatment.

Fungicidal activity of peptides encoded by immunoglobulin genes.

Evidence from previous works disclosed the antimicrobial, antiviral, anti-tumour and/or immunomodulatory activity exerted, through different mechanisms of action, by peptides expressed in the complementarity-determining regions or even in the constant region of antibodies, independently from their specificity and isotype. Presently, we report the selection, from available databases, of peptide sequences encoded by immunoglobulin genes for the evaluation of their potential biological activities. Synthetic peptides representing the translated products of J lambda and J heavy genes proved to act in vitro against pathogenic fungi, entering yeast cells and causing their death, and exerted a therapeutic effect in a Galleria mellonella model of infection by Candida albicans. No haemolytic, cytotoxic and genotoxic effects were observed on mammalian cells. These findings raise the hypothesis that antibodies could be the evolutionary result of the adaptive combination of gene products ancestrally devoted to innate antimicrobial immunity.

A Biotechnological Approach for the Development of New Antifungal Compounds to Protect the Environment and the Human Health.

BACKGROUND: In the Po Valley aflatoxins play a relevant role: the local food economy is heavily based on cereal cultivations for animal feed and human nutrition. Aims of this project are the identification of new compounds that inhibit Aspergillus proliferation, the development of new inhibitors of aflatoxins production, and the set-up a practical screening procedure to identify the most effective and safe compounds. DESIGN AND METHODS: New compounds will be synthetized with natural origin molecules as ligands and endogenous metal ions to increase their bioavailability for the fungi as metal complexes. A biotechnological high-throughput screening will be set up to identify efficiently the most powerful substances. The newly synthesized compounds with effective antifungal activities, will be evaluated with battery of tests with different end-points to assess the toxic potential risk for environmental and human health. EXPECTED IMPACT OF THE STUDY FOR PUBLIC HEALTH: The fundamental step in the project will be the synthesis of new compounds and the study of their capability to inhibit aflatoxin biosynthesis. A new, simple, inexpensive and high-throughput method to screen the anti-fungine and anti-mycotoxin activity of the new synthesised compounds will be applied. The evaluation of possible risks for humans due to toxic and genotoxic activities of the molecules will be made with a new approach using different types of cells (bacteria, plants and human cells). Significance for public healthAflatoxins contamination constitutes a health emergency because aflatoxins and mycotoxins, besides being toxic, are among the most carcinogenic substances known. Even if Aspergillus are dominant in tropical regions, recently are becoming a serious problem also in Europe and in Italy, especially in area as the Po Valley in which this problem play a particularly important role, because the local food economy is heavily based not only on cereal cultivations aimed at animal feed but also on the production of derivatives to human nutrition. The aims of this research are the development of new bioactive molecules, obtained by natural molecules and metal ions, that are able to reduce the risk of food contamination by aflatoxin, but are harmless for environmental and health and the evaluation of the newly synthesized compounds using a battery of tests with different end-points to assess the toxic potential risk for environmental and human health.

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

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

Peptides of the constant region of antibodies display fungicidal activity.

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

Anti-proliferative activity and chemoprotective effects towards DNA oxidative damage of fresh and cooked Brassicaceae.

Epidemiological evidence shows that regular consumption of Brassicaceae is associated with a reduced risk of cancer and heart disease. Cruciferous species are usually processed before eating and the real impact of cooking practices on their bioactive properties is not fully understood. We have evaluated the effect of common cooking practices (boiling, microwaving, and steaming) on the biological activities of broccoli, cauliflower and Brussels sprouts. Anti-proliferative and chemoprotective effects towards DNA oxidative damage of fresh and cooked vegetable extracts were evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and Comet assays on HT-29 human colon carcinoma cells. The fresh vegetable extracts showed the highest anti-proliferative and antioxidant activities on HT-29 cells (broccoli>cauliflower = Brussels sprouts). No genotoxic activity was detected in any of the samples tested. The cooking methods that were applied influenced the anti-proliferative activity of Brassica extracts but did not alter considerably the antioxidant activity presented by the raw vegetables. Raw, microwaved, boiled (except broccoli) and steamed vegetable extracts, at different concentrations, presented a protective antioxidative action comparable with vitamin C (1 mm). These data provide new insight into the influence of domestic treatment on the quality of food, which could support the recent epidemiological studies suggesting that consumption of cruciferous vegetables, mainly cooked, may be related to a reduced risk of developing cancer.

Cancer treatment-induced cardiotoxicity: a cardiac stem cell disease?

Cardiovascular diseases and cancer represent respectively the first and second cause of death in industrialized countries. These two conditions may become synergistic when cardiovascular complications of anti-cancer therapy are considered. More than 70% of childhood and 50% of adult cancer patients can be cured, however this important success obtained by the biological and medical research is obfuscated by emerging findings of early and late morbidity due to cardiovascular events. Although anthracyclines are effective drugs against cancer a dose-dependent cardiotoxic effects whose mechanism has not been elucidated resulting in failure of therapeutic interventions limit their use. Unexpectedly, tyrosine/kinase inhibitors (TKIs) aimed at molecularly interfering with oncogenic pathways, have been implicated in cardiac side effects. Possible explanations of this phenomenon have been ambiguous, further strengthening the need to deepen our understanding on the mechanism of cardiotoxicity. In addition to a detailed description of anthracyclines and TKIs-related cardiovascular effects, the present review highlights recent observations supporting the hypothesis that the cellular target of anthracyclines and TKIs may include myocardial compartments other than parenchymal cells. The demonstration that the adult mammalian heart possesses a cell turnover regulated by primitive cells suggests that this cell population may be implicated in the onset and development of cardiovascular effects of anti-cancer strategies. The possibility of preventing cardiotoxicity by preservation and/or expansion of the resident stem cell pool responsible for cardiac repair may open new therapeutic options to unravel an unsolved clinical issue.

Genotoxicity revaluation of three commercial nitroheterocyclic drugs: nifurtimox, benznidazole, and metronidazole.

Nitroheterocyclic compounds are widely used as therapeutic agents against a variety of protozoan and bacterial infections. However, the literature on these compounds, suspected of being carcinogens, is widely controversial. In this study, cytotoxic and genotoxic potential of three drugs, Nifurtimox (NFX), Benznidazole (BNZ), and Metronidazole (MTZ) was re-evaluated by different assays. Only NFX reduces survival rate in actively proliferating cells. The compounds are more active for base-pair substitution than frameshift induction in Salmonella; NFX and BNZ are more mutagenic than MTZ; they are widely dependent from nitroreduction whereas microsomal fraction S9 weakly affects the mutagenic potential. Comet assay detects BNZ- and NFX-induced DNA damage at doses in the range of therapeutically treated patient plasma concentration; BNZ seems to mainly act through ROS generation whereas a dose-dependent mechanism of DNA damaging is suggested for NFX. The lack of effects on mammalian cells for MTZ is confirmed also in MN assay whereas MN induction is observed for NFX and BNZ. The effects of MTZ, that shows comparatively low reduction potential, seem to be strictly dependent on anaerobic/hypoxic conditions. Both NFX and BNZ may not only lead to cellular damage of the infective agent but also interact with the DNA of mammalian cells.