Jaouad Anter

Antigenotoxicity, Cytotoxicity, and Apoptosis Induction by Apigenin, Bisabolol, and Protocatechuic Acid

Medicinal plants represent an important resource in new drug research. Antioxidant properties of plants can help to scavenge reactive oxygen species. The objective of this work was to evaluate the genotoxic, antigenotoxic, tumoricidal, and apoptotic effect of some major phenols (apigenin, bisabolol, and protocatechuic acid) from two medicinal plants, Matricaria chamomilla and Uncaria tomentosa. The wing spot test of Drosophila melanogaster was used to evaluate the genotoxicity and antigenotoxicity of the three phenols. The human model of HL-60 leukemia cells was used for the assessment of the cytotoxic effect, growth, and cellular viability. The apoptotic effect was evaluated using a DNA fragmentation assay based on the formation of internucleosomal units. Protocatechuic acid (0.25 and 1 mM), apigenin (0.46 and 1.85 mM), and bisabolol (0.56 and 2.24 mM) did not exhibit any genotoxic effect. The three phenols showed an antigenotoxic effect against the hydrogen peroxide effect and also exhibited tumoricidal activity. Apigenin (2.24-35.96 mM) showed a lower 50% inhibitory concentration (0.75 and 3.87 mM for the trypan blue test and WST-8 colorimetric assay, respectively) than bisabolol and protocatechuic acid. These phenolics also induced apoptosis in HL-60 leukemia cells. This study suggests that the antioxidant activity of Chamomilla and Uncaria could be partially responsible of their beneficial activity.

A pilot study on the DNA-protective, cytotoxic, and apoptosis-inducing properties of olive-leaf extracts.

Leaves of olive trees are an abundant raw material in the Mediterranean basin. They contain large amounts of potentially useful phytochemicals and could play beneficial roles in health care. In the present study, the principal bioactive phenols in olive-leaf extracts (OLEs) have been identified and quantified, and their genotoxic/antigenotoxic, cytotoxic and apoptotic effects have been assessed. The Somatic Mutation and Recombination Test (SMART) in wing imaginal discs of Drosophila melanogaster has been performed to test the possible genotoxicity of overall OLE and the individual components oleuropein and luteolin at different concentrations. The same assay was able to detect antigenotoxic activity against hydrogen peroxide as oxidative genotoxicant. None of the extracts/phenols tested showed significant mutagenic activity. This fact, together with the antigenotoxic activity against H(2)O(2) detected for all these extracts/phenols, confirmed the safety of OLE, oleuropein and luteolin in terms of DNA protection. HL60 human promyelocytic leukemia cells were used to assess the cytotoxic effects of the extracts/phenols. OLE, oleuropein and luteolin showed a dose-dependent cytotoxic effect with different IC50 (10μl/ml, 170μM, and 40μM, respectively). DNA fragmentation patterns and cell staining with acridine orange and ethidium bromide indicated that the mechanism for the cytotoxic effect of OLE, oleuropein and luteolin was the apoptotic pathway, with DNA laddering and cytoplasmic and nuclear changes. These results could help explain the mechanism of action that underlies the beneficial effect of OLE, proposed as a nutraceutical in the prevention of human cancer.

Modulation of genotoxicity and cytotoxicity by radish grown in metal-contaminated soils

Members of the Brassicaceae family are known for their anticarcinogenic and genetic material protective effects. However, many of the species of this family accumulate high amounts of metals, which is an undesirable feature. Radish (Raphanus sativus L.) has shown to accumulate metals in roots to a higher extent than others members of Brassicaceae. The main objectives of this work are (i) to study the distribution of the accumulated As, Pb and Cd in radish plants and (ii) to establish the genotoxic, antigenotoxic and cytotoxic activities of the root and shoot of this vegetable. Results indicate that (i) the shoots of radish accumulate higher concentrations of metal(oid)s than roots; (ii) the shoots were genotoxic at the different concentrations studied, with the root showing such genotoxic effect only at the highest concentration assayed; (iii) the antigenotoxic potential of radish is reduced in plants with high metal content and (iv) the tumouricide activities of the radish plants were negatively correlated to their metal(oid) contents. An interaction between metal(oid)s and the isotyocianates (hydrolysis products of the glucosinolates) contained in the radish is suggested as the main modulator agents of the genotoxic activity of the plants grown in contaminated soils with metal(oid)s.

Role of Citrus Juices and Distinctive Components in the Modulation of Degenerative Processes: Genotoxicity, Antigenotoxicity, Cytotoxicity, and Longevity in Drosophila

It is well established that breakfast beverages contain high quantities of Citrus juices. The purpose of the present study was to assess the nutraceutical value of orange and lemon juices as well as two of their active compounds: hesperidin and limonene. Indicator assays were performed at three levels to evaluate different biological health promoter activities: (i) determination of the safety and DNA-damage protecting ability against free radicals by using the somatic mutation and recombination test (SMART) in Drosophila melanogaster, (ii) study of the modulating role for life span in Drosophila melanogaster, and (iii) measurement of the cytotoxic activity against the human tumor cell line HL60. The highest concentrations assayed for lemon juice and limonene (50% v/v and 0.73 mM, respectively) showed genotoxic activity as evidenced from SMART. Orange and lemon juices as well as hesperidin and limonene exhibit antigenotoxic activity against hydrogen peroxide used as an oxidative genotoxin. Life-span experiments revealed that the lower concentrations of orange juice, hesperidin, and limonene exerted a positive influence on the life span of Drosophila. Finally all substances showed cytotoxic activity, with hesperidin being least active. Taking into account the safety, antigenotoxicity, longevity, and cytotoxicity data obtained in the different assays, orange juice may be a candidate as a nutraceutical food as it (1) is not genotoxic, (2) is able to protect DNA against free radicals, and (3) inhibits growth of tumor cells.

Effects of oocyte quality, incubation time and maturation environment on the number of chromosomal abnormalities in IVF-derived early bovine embryos

Chromosomal aberrations are one of the major causes of embryo developmental failures in mammals. The occurrence of these types of abnormalities is higher in in vitro-produced (IVP) embryos. The aim of the present study was to investigate the effect of oocyte morphology and maturation conditions on the rate of chromosomal abnormalities in bovine preimplantational embryos. To this end, 790 early cattle embryos derived from oocytes with different morphologies and matured under different conditions, including maturation period (24 v. 36h) and maturation media (five different serum supplements in TCM-199), were evaluated cytogenetically in three sequential experiments. The rates of normal diploidy and abnormal haploidy, polyploidy and aneuploidy were determined in each embryo. Throughout all the experiments, the rate of chromosomal abnormalities was significantly (P<0.05) affected by oocyte morphology and maturation conditions (maturation time and culture medium). Lower morphological quality was associated with a high rate of chromosome abnormalities (P<0.05). Moreover, polyploidy was associated with increased maturation time (P<0.01), whereas the maturation medium significantly (P<0.05) affected the rates of haploidy and polyploidy. In general, supplementing the maturation medium with oestrous cow serum or fetal calf serum resulted in higher rates of chromosomal aberrations (P<0.05) compared with the other serum supplements tested (bovine steer serum, anoestroues cow serum, bovine amniotic fluid and bovine serum albumin). On the basis of the results of the present study, we conclude that the morphological quality of oocytes and the maturation conditions affect the rate of chromosomal abnormalities in IVP bovine embryos.

Transcriptomic Analyses of Adipocyte Differentiation From Human Mesenchymal Stromal-Cells (MSC)

Adipogenesis is a physiological process required for fat‐tissue development, mainly involved in regulating the organism energetic‐state. Abnormal distribution‐changes and dysfunctions in such tissue are associated to different pathologies. Adipocytes are generated from progenitor cells, via a complex differentiating process not yet well understood. Therefore, we investigated differential mRNA and miRNA expression patterns of human mesenchymal stromal‐cells (MSC) induced and not induced to differentiate into adipocytes by next (second)‐generation sequencing. A total of 2,866 differentially expressed genes (101 encoding miRNA) were identified, with 705 (46 encoding miRNA) being upregulated in adipogenesis. They were related to different pathways, including PPARG, lipid, carbohydrate and energy metabolism, redox, membrane‐organelle biosynthesis, and endocrine system. Downregulated genes were related to extracellular matrix and cell migration, proliferation, and differentiation. Analyses of mRNA‐miRNA interaction showed that repressed miRNA‐encoding genes can act downregulating PPARG‐related genes; mostly the PPARG activator (PPARGC1A). Induced miRNA‐encoding genes regulate downregulated genes related to TGFB1. These results shed new light to understand adipose‐tissue differentiation and physiology, increasing our knowledge about pathologies like obesity, type‐2 diabetes and osteoporosis. J. Cell. Physiol. 232: 771–784, 2017.

Demethylating and anti‐hepatocarcinogenic potential of hesperidin, a natural polyphenol of Citrus juices

Hepatocellular carcinoma (HCC) is a neoplasia representing the fifth most common malignancy worldwide and the third cause of death from cancer. Diets with high content in fruits and vegetables are widely recommended for their health‐promoting properties, among them, the protection against diabetes, cancer, and cardiovascular diseases. Hesperidin is the most important phenol in the orange fruit with well‐known health benefits. Diet components have been used as possible modulator agents of DNA methylation in cancer cells and epigenetic therapy against their harmful effects could be a potential tool in chemotherapy. The purpose of the present study was to evaluate the methylation patterns induced by hesperidin in HL60 cell line as an in vitro model in order to analyze its chemopreventive effects in epigenetic cancer therapies. A parallel in vivo pilot experience using a rat diethyl nitrosamine hepatocarcinogenesis‐induced model was carried out to validate the therapeutic efficacy of this orange flavonol. Results showed that: (i) Hesperidin is cytotoxic in a dose‐dependent manner and the IC50 was 12.5 mM; (ii) Hesperidin exerts a significant hypomethylating effect on the LINE‐1 sequence (up to 47% hypomethylation at 12.5 mM) and on the ALU‐M2 repetitive sequences (up to 32% at 6 mM) in HL60 tumor cells. (iii) Hesperidin does not affect the rat body and liver weight and it is able to reduce the diethyl nitrosamine‐induced nodules at 1,000, 500, and 250 ppm. In conclusion, hesperidin could be proposed as a candidate molecule in chemoprevention in epigenetic therapy purposes.

Anti/genotoxic, longevity inductive, cytotoxic, and clastogenic-related bioactivities of tomato and lycopene: Tomato and Lycopene Bioactivities

The aim of this study was to evaluate some biological activities of tomato as well as lycopene and to consider a new nutraceutic value for this fruit regarding to the protection against genetic damage and as a chemopreventive agent. Genotoxicity, DNA‐protection against hydrogen peroxide, and lifespan properties of tomato and lycopene were assessed through wing spot test and longevity assay using the Drosophila in vivo model. Additionally, chemopreventive activity was investigated through cytotoxicity, DNA‐fragmentation comet and annexin V FITC/PI assays using HL60 in vitro model. Results showed that: (i) tomato and lycopene are not genotoxic and protect against H2O2‐induced damage; (ii) with respect to the lifespan, tomato and lycopene are harmless at the lowest concentration; (iii) tomato is cytotoxic in a dose‐dependent manner, but not lycopene; (iv) tomato and lycopene do not induce internucleosomal DNA‐fragmentation although they induce significant clastogenic activity at low level in the leukemia cells. To sum up, tomato is a good candidate to be considered as a nutraceutical substance. Furthermore, synergistic action among other components within tomato matrix could be the cause of the health effects observed in this vegetable, which are not fully explained by lycopene. Environ. Mol. Mutagen. 59:427–437, 2018.