Analysis of delphinidin and luteolin genotoxicity in human lymphocyte culture

Introduction: Biofl avonoids delphinidin (2-(3,4,5-Trihydroxyphenyl)chromenylium-3,5,7-triol) and luteolin (2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-chromenone) have been recognized as promising antioxidants and anticancer substances. Due to their extensive use, the goal of the research was to determine whether they have any genotoxic potential in vitro. Methods: Analysis of genotoxic potential was performed applying chromosome aberrations test in human lymphocyte culture, as this kind of research was not conducted abundantly for these two biofl avonoids. Delphinidin and luteolin were dissolved in DMSO and added to cultures in fi nal concentrations of 25, 50 and 100 μM. Results: In human lymphocytes cultures Delphinidin induced PCDs in all treatments, potentially affecting the cell cycle and topoisomerase II activity. In concentration of 50 μM luteolin showed strong genotoxic effects and caused signifi cant reduction of cell proliferation. Conclusion: Luteolin exhibited certain genotoxic and cytostatic potential. Delphinidin was not considered genotoxic, however its impact on mitosis, especially topoisomerase II activity, was revealed.


INTRODUCTION
Delphinidin and luteolin belong to the group of fl avonoids.Th ey are plant pigments mostly found in fl owers and fruits, consumed on a daily basis.Antioxidative eff ects of biofl avonoids have been proved in many biological studies, meaning that they are recognized as very effi cient natural protectants.
Delphinidin is abundantly present in the fl owers and fruits of the following: plum, grapes, currant, blueberry, cranberry, strawberry, raspberry, blackberry, elderberry (1,2).It has been proven that delphinidin has a protective role and it decreases the micronuclei frequency in vivo (3).It also shows cytostatic eff ects in a concentration-dependent gradient (4), antiangiogenic eff ects in tumor tissue (5), and the ability to induce apoptosis in cancer cells (6).Th e genotoxic eff ects of delphinidin have not been completely explored.Results of the Ames test suggest a general fl avonoid genotoxicity (7), but specifi c analysis of delphinidin show that it does not manifest genotoxic eff ects (3).It is also been noticed that delphinidin can inhibit the activity of topoisomerase II, that plays a role in chromosome segregation during mitosis (2).
Luteolin is a plant fl avonoid from fl avone class.It is a polyphenolic compound, with certain pharmaceutical characteristics, found in various fruits, vegetables, seeds.Th e clinical studies aiming to assay the anticancer eff ects of diff erent biofl avonoids according to their antioxidant potential, suggest luteolin as a potential inhibitor of cell proliferation (8).In the cells of various cancer types, luteolin is proven to be an eff ective inhibitor of cell proliferation in the average range of concentrations from 3-50 microns (9).Luteolin, like some other fl avonoids, can stop the cell cycle of cancer cells at G1/S or G2/M checkpoints (10).It has been proven that luteolin can stop the cell cycle in G1 phase in the cells of human melanoma by inhibiting the activity of CDK2 (cyclin-dependent kinase 2), the enzyme that participates in progression of the cell cycle (11).Luteolin poses the potential to inhibit an angiogenesis by the suppression of the angiogenic factor VEGF (vascular endothelial factor) expression in the cancer cells.Th e antimetastatic eff ects of luteolin can be attributed to the suppression of the cytokines synthesis, such as TNFα (tumor necrosis factor α) and IL-6 (interleukin 6) involved in tumor cells migration and metastasis (10).
Regarding presented fl avonoids bioactivity, the goal of this research was to determine the genotoxicity of delphinidin and luteolin in human lymphocyte cultures of peripheral blood using chromosome aberrations analysis.

Tested substances
Delphinidin is an anthocyanin, and has the molecular mass of 338.69664 g/mol, with the molecular formula C 15 H 11 O 7 + .Delphinidin is a pigment, whose color varies from a purple-blue shade (pH 6-7) to a bright red shade (pH 1-3).
Luteolin is a fl avone, and has the molecular mass of 286.2363 g/mol, with the molecular formula C 15 H 10 O 6 .Luteolin is a common plant pigment, whose color is yellow.

Chromosome aberrations analysis
Human lymphocyte cultures of 4 donors (2 ♂ i 2 ♀), healthy non-smokers of approximately the same age, were established immediately upon venipuncture of the cubital vein, in sterile vacutainers containing sodium heparin (BD Vacutainer Systems, Plymonth, UK).All participants in the study had signed the informed consent.
Cultures were set up by addition of 400 μl of whole blood in 5 ml of PBMAX TM Karyotyping Medium (GIBCO-Invitrogen, Carlsbad, CA, USA).Incubation lasted for 72h on 37°C (Cytoperm 8080, Heraeus, Germany).Th e cell division was blocked in metaphase by the colcemid treatment in the concentration of 0.18 μg/ml 90 minutes before the cell harvesting.Cell harvesting included hypotonic (0.75% KCl) treatment followed by centrifugation (1000 rpm for 10 minutes) and tripled of ice-cold acetic-alcohol fi xative treatments and centrifugations.Cell suspension was dropped on ice-cold coded slides.Air-dried microscopic preparations were stained in 5% Giemsa stain in Gurr buff er (GIBCO-Invitrogen, Carlsbad, CA, USA).
Slides were analyzed on an Olympus BX51 microscope, on 1000x magnifi cation.Analysis included observation of structural and numerical chromosome aberrations according to the International System for Human Cytogenetic Nomenclature.Structural aberrations were classifi ed as: aberrations of chromosomal (chr) type (chrb-chromosome breaks, ace-acentric fragments), and aberrations of chromatid (cht) type (chtb-chromatid breaks) (14).
Since the reduction in metaphase spreads of cultures treated with luteolin was noticed, these slides were additionally used to determine mitotic activity expressed as mitotic index (MI).

Statistical analysis
Th e mean, standard deviation, standard error of the mean, and variability coeffi cient were calculated using Microsoft Excel 2007.Proportion comparison (Z-test), using Winks 4.5 Professional edition (TexaSoft, Cedar Hill, Texas) was applied to determine signifi cance of diff erences between treatments and controls.

RESULTS
Th e most common of the registered aberrations in delphinidin treated cultures were PCD (premature centromere division).At 100 μM, there was a PCD registered in each of 4 samples with the signifi cant diff erence in comparison against controls (z=-2.005;p=0.045).
Summarized results of chromosome aberrations analysis in 400 metaphases (4 lymphocyte sample cultures) of controls and delphinidin treated cultures are presented in Table 1.Relative frequencies of observed chromosome aberrations in controls and lymphocytes cultures treated with tested concentrations of delphinidin are shown in Figure 1.
Summarized results of chromosome aberrations analysis of controls and luteolin treated cultures are presented in Table 2. Relative frequencies of observed chromosome aberrations in controls and lymphocytes cultures treated with tested concentrations of luteolin are shown in Figure 2. Discussion Th e results of the chromosome aberration analysis and the associated statistical analysis have shown that delphinidin in tested concentrations does not signifi cantly increase observed categories of aberrations, except PCD.Th ese results are completely concordant with the previous research on delphinidin genotoxicity confi rming that delphinidin is not genotoxic, even in extremely high concentrations    (3,15,16).Opposing, it has been reported that delphinidin has a strong cytotoxic and cytostatic eff ects, especially in cancer cells (3)(4)(5)(6).Although the mechanism of PCD has not been completely described, it is considered that the inhibition of topoisomerase II may be the basis and cytostatics are recognized as the main cause of PCD (17).Also, the significant increase of PCD frequencies is being associated with cytotoxic eff ect of delphinidin, assuming that delphinidin induces premature centromere division by inhibiting topoisomerase II.Playing the significant role in chromosome segregation during mitosis, topoisomerase II induces endoreduplication.Luteolin and delphinidin treatments of human lymphocyte cultures were previously reported to induce endoreduplications in the presence of halogenated boroxine (18).Also, the potential to inhibit the topoisomerase II activity was previously confi rmed for luteolin (12).However, in the presented research, the most significant eff ect of luteolin in human lymphocytes culture was inhibition of cell proliferation.It is known that luteolin is an eff ective inhibitor of some cancer cell proliferation and is also able to arrest the cell cycle in G1/S and G2/M checkpoints (10).In the concentration of 50 μM luteolin inhibits genotoxic eff ects induced by halogenated boroxine and reduce cell proliferation in vitro (18).Determined signifi cant increase of structural chromosome aberrations as well as aneuploidies for lymphocytes cultures treated with luteolin in concentration of 50 μM, presents the important fi nding as chromosome aberrations are the primary genotoxicity biomarker associated with the increased cancer risk (19).Th e reduction of the mitotic activity could be the consequence of DNA synthesis inhibition or blocking of the cell cycle in G phase (20,21).

CONCLUSION
Chromosome aberrations analysis of selected biofl avonoids in tested concentrations applied in human lymphocyte cultures has revealed that delphinidin is neither clastogenic nor aneugenic but the incidence of PCDs may indicate its impact on mitosis and especially topoisomerase II activity.However, luteolin exhibits genotoxic eff ects in concentration of 50 μM while the most considerable eff ect of luteolin is the reduction of cell proliferation revealing its remarkable cytostatic potential.

TABLE 1 .
Results of chromosome aberrations analysis upon delphinidin treatment