THE GENOTOXICITY OF VITAMIN C In vitro

Th e genotoxic eff ects of Vitamin C (ascorbic acid) on human lymphocytes in vitro were estimated by analyzing and identifying various chromosome abnormalities, in relation to the concentration of Vitamin C. Testing concentrations of Vitamin C induced diff erent aberrations including the impairment of spindle function. Th e spindle disturbances can result in mitotic arrest, multipolar spindles and multipolar segregation, errors in chromosome segregation, formation of chromosome bridges and chromosome laggards. Th e most frequent irregularities were found in anaphase and telophase. A certain number of lymphocytes were arrested at anaphase or telophase (in colchicine-untreated cultures of human lymphocytes). Testing concentrations of ascorbic acid did not induce a signifi cant increase in the number of aneuploid mitoses and were not clastogenic except at the highest concentration (, μg/ml) in colchicine-treated cultures, and in colchicine-untreated cultures of human lymphocytes the pulverization of chromosome was observed. Vitamin C changed the mitotic index value of lymphocytes notably at the higher concentrations (,  and , μg/ml).


Introduction
Th e great number of previous investigations indicate that Vitamin C is an antioxidant and a strong free-radical scavenger (, , ). Th is vitamin can protect cellular macromolecules, including DNA, from oxidative damage induced by diff erent agents. Besides the antimutagenic and anticlastogenic properties of Vitamin C, it is also known that this vitamin has mutagenic and clastogenic eff ects. Shamberger () summarized the genotoxic eff ects of Vitamin C in diff erent test systems. Vitamin C possesses mutagenic activity inducing DNA strand breakage and chromosome aberrations, increasing the frequency of sister-chromatid exchanges (SCEs) in cells and increasing the number of somatic mutations. However, Vitamin C is not an active mutagen itself. Oxy radicals, appearing during the oxidation of the vitamin, are implicated in toxic actions of Vitamin C in vitro. Nowhere in the experiments in vivo did Vitamin C display genotoxicity. All of the mutagenic eff ects of this vitamin were demonstrated in tests in vitro. Th is may be due to the presence of an antioxidant system which inactivates oxy radicals before their interaction with DNA, and due to the presence of a spatial separation of the components of oxy-radical generating system in living organisms.
In some cases Vitamin C acts as a comutagen by enhancing the gene mutations, SCEs and chromosome aberrations induced by different chemicals. It enhanced the mutagenicity at high concentrations, whereas at lower concentrations it decreased the frequency of induced aberrations by diff erent chemicals. Vitamin C reduced the clastogenic effects of many agents, decreased the number of SCEs, inhibited the gene mutations and decreased the frequency of micronuclei formation. At lower concentrations, it is probable that the process of O-elimination by Vitamin C was predominant, but at higher concentrations Vitamin C displayed its own genotoxic properties. As an antioxidant, Vitamin C may also have a potential anticarcinogenic activity, but its possible role and mechanism of action in cancer prevention has not been clearly established. In this study, the genotoxicity of different concentrations of Vitamin C on human peripheral blood lymphocytes in cultures have been estimated by the analysis of chromosome aberrations in standard cytogenetics test in colchicinetreated cultures and by the analysis of genotoxic effects of Vitamin C at all phases of the mitotic process in colchicine-untreated cultures of human lymphocytes.

Materials and Methods
Vitamin C (Pliva, Zagreb) or ascorbic acid was used to investigate eff ects of this vitamin on the genetic material of cultured human lymphocytes. Medium RPMI-, Fetal calf serum (FCS), Phytohemagglutinin (PHA) were purchased from Sigma Chemical Co., St. Louis, MO., Colchicine (COL) from Kemika, Zagreb. Th e test of chromosome aberrations was modifi ed according to Moorhead et al. (). Th e study was performed on the peripheral blood lymphocytes obtained from six healthy donors, three females and three males aged  to  years. Th e heparinised human blood was cultured in medium RPMI- and supplemented with  fetal calf serum. Th e lymphocytes were stimulated to divide by adding phytohemagglutinin ( μg/ml). All cultures were incubated at °C for a total  hours in the dark. Vitamin C was added to lymphocyte cultures after  hours of incubation at following fi nal concentrations: , , , , ,  and  μg/ml culture medium and the cultures incubated for a further  hours. Vitamin C was dissolved in sterile water just before each experiment. Th ese concentrations of Vitamin C were chosen on the basis of published data (, ) and preliminary tests. Two cultures were prepared simultaneously for each concentration of Vitamin C: one culture was without colchicines, enabling the analysis of genotoxic eff ects of Vitamin C at all phases of the mitotic process, and in the other culture cells were arrested in metaphase by the addition of colchicine at a fi nal concentration of , μg/ml culture medium two hours before harvesting for cytogenetic analysis of metaphases karyotype. Th e control, untreated cultures of lymphocytes, were established as well. After hypotonic treatment with , mol/dm KCl for  min at  °C, cells were gently fi xed four times with freshly prepared fi xative ( parts methanol:  part glacial acetic acid) at room temperature. Th e cells were harvested by centrifuging. A small amount of cell suspension was drawn into a Pasteur pipette and, as normally done, three drops were expelled carefully on the slides and air-dried. Five to six slides were made from each culture. Th e chromosome preparations were stained for  min in  Giemsa. Data for calculating the value of the mitotic index were obtained by analyzing the number of mitotic cells per  cells of a culture, i.e. per  cells for each concentration of Vitamin C and control. In order to obtain the frequencies of chromosomal aberrations,  metaphases from each colchicine-treated culture of one subject were examined for a total of  cells for each concentration of Vitamin C and control. But, for Vitamin C concentrations of ,  and  μg/ml were analyzed  cells and  metaphases for each concentration of Vitamin C and control because of a small number of cells in mitosis for analysis. Numerical and structural chromosome aberrations were analyzed separately. In colchicine-untreated cultures of human lymphocytes, the genotoxic activity of Vitamin C was studied scoring  dividing cells for each concentration of Vitamin C and control for the frequency of anaphase/telophase cells and the frequency of mitotic cells with different aberrations. The significance of the value of the mitotic index, the number of metaphases with numerical and structural chromosome aberrations and the number of cells at anaphase/telophase and the number of cells with different aberrations induced by Vitamin C in comparison with the control cultures were analyzed using the chi-square test (χ-test).

Results
Analysis of chromosome aberrations in standard cytogenetics test in colchicine-treated cultures showed that, in comparison with the control cultures, treatments with Vitamin C at the test concentrations of , ,  and  μg/ml culture medium, did not change the mitotic index value of lymphocytes notably ( Table  ). The mitotic index value of human lymphocytes treated with Vitamin C concentrations of ,  and  μg/ml culture medium was signifi cantly reduced (statistically significant at  μg/ml: p < ,; at  μg/ml: p < , and at  μg/ml: p < ,) ( Table ). Th e number of abnormal metaphases with numerical and structural chromosome aberrations (chromosome and chromatid breaks, chromosome rearrangements and acentric fragments) was not statistically significant in treated cultures when compared with that of the control except in culture treated with  μg/ml Vitamin C (Table . and Table ). Th e highest testing concentration of Vitamin C ( μg/ml) in the presence of colchicine had clastogenic effect (p < ,). This vitamin did not induce aneuploidy and polyploidy remarkably excluding at the highest concen-tration of Vitamin C (, μg/ml: p < ,). Endoreduplicated chromosomes (chromosomes which replicate but cannot divide correctly) have been noted in lymphocyte cultures after treatments with Vitamin C. Analysis of genotoxic eff ects of Vitamin C at all phases of the mitotic process in colchicine-untreated lymphocyte cultures of human lymphocytes showed that effects of this vitamin already became evident after treatment with the concentration of  μg/ml, and reached a maximum at the concentration of  μg/ml (Table ). In lymphocyte cultures treated in vitro with Vitamin C, but not treated with colchicine, a certain number of mitoses were arrested at anaphase or telophase when compared with the control culture (Table ). Th e photomicrographs of human lymphocytes arrested at anaphase-telophase are shown in Figure    Th e total number of aberrant phases 0 7** 33*** 16*** 14*** 11*** 9** 8**

Aberrant anaphases and telophases
No.  (Table ). Such an increase was particularly remarkable in lymphocytes treated with  μg/ml Vitamin C (p < ,). Th e predominant aberration was irregular kinetics of chromosomes during anaphase and telophase as is shown in Table III (statistically significant at  μg/ml: p < , and at  μg/ml: p < , to the total number of aberrant phases) and Figure . Th e frequency of aberrant anaphases and telophases at all concentrations exceed over  of the total number of aberrant cells. Th e aberrant anaphases contained lagging chromatin, including both whole chromatids and chromosomes lagging behind between the poles (Figure c), multipolar anaphases (tripolar and tetrapolar) with disrupted poles and unequal distribution of chromosomes (Figure d) and chromosome bridges stretching between the poles (Figure e). Some chromosomes or a group of chromosomes may be apart in relation to the main groups of chromosomes (Figure c). Similar aberrations were found at telophase cells ( Figure f). Also, the frequent types of mitosis-disruptive changes were chromosome adhesiveness and stickiness (Figure e). The results of the cytogenetic analysis of metaphase cells showed that the aberrant metaphases involved dislocation of whole chromosomes or groups of chromosomes from metaphase plate. A pulverization of chromosomes (the total disintegration of chromosomes) has been reported after treatments with Vitamin C in colchicine-untreated lymphocyte cultures.

Discussion
Th e results obtained from the chromosome aberrations analysis indicate that the genotoxicity of Vitamin C in human lymphocyte cultures varies depending on concentration. Vitamin C had the most heterogeneous genotoxic eff ect at the concentration of  μg/ml and most effi cient genotoxic eff ects at the concentrations of ,  and  μg/ml. Th e testing concentrations of Vitamin C induced various aberrations including the impairment of spindle function. Th e spindle disturbances can result in mitotic arrest, multipolar spindles, errors in chromosome segregation, chromosome bridges and chromosome laggards. Th e most frequent irregularities were found at anaphase and telophase. Th e testing concentrations of ascorbic acid were not clastogenic except at the highest concentration ( μg/ml) in colchicinetreated cultures, and pulverization of chromosome was observed in colchicine-untreated cultures indicating that certain higher concentrations of ascorbic acid have clastogenic eff ects. Th e results obtained by Antunes and Takahashi () showing that ascorbic acid at concentration of  μg/ml produced clastogenic eff ects in human lymphocyte cultures also confi rmed this possibility. Th e treatments with Vitamin C (, ,  and  μg/ ml) did not signifi cantly change the mitotic index value of lymphocytes in the cytogenetics test of chromosome aberrations. Th is indicates that Vitamin C did not inhibit cell proliferation at these testing concentrations. Th e results of many in vitro studies have indicated that Vitamin C can be mutagenic (clastogenic) and cytotoxic. Vitamin C at high concentrations possesses genotoxic properties towards Chinese hamster ovary (CHO) cells: induced sister chromatid exchanges () and chromosome aberrations (). Exposure of separated lymphocytes in vitro to Vitamin C, at doses greater than  μmol/ dminduced strand breakage (). Vitamin C produced a dose-related increase in DNA damage (). Ascorbate has caused increase in SCEs in human lymphocytes (). Contrary to in vitro studies, vitamin C did not induce SCEs in the bone marrow cells of Chinese hamsters (), was not cytotoxic and did not inhibit proliferation of lymphocytes in cultures (). Vitamin C, in vitro, successfully reduced the clastogenic eff ect of many antitumour agents such as cyclophosphamide (), doxorubicin (, , ), cisplatin () and bleomycin (, ). It also has in vivo anticlastogenic eff ect against chromosomal damage and the number of abnormal metaphases induced by antitumour agents such as cisplatin in rodents (, , , ) and induced by cyclophosphamide (). Ascorbic acid also reduces the chromosome aberrations induced by ethyl methanesulfonate (). It decreases the number of SCEs induced by cyclophosphamide (), mitomycin C () and oxygen radicals () and inhibits the gene mutations induced by ethyl methane sulfonate (, ) and cisplatin (). Vitamin C decreases the frequency of micronucleus formation induced by cisplatin (), bleomycin () and doxorubicin (). Sometimes, Vitamin C acted as a comutagen and increased HOand bleomycin-induced chromosomal aberrations (). In combination with HO there were small protective eff ects at low doses and exacerbating eff ects at high doses () towards DNA damage in human lymphocytes. According to Park () and Osmak et al. () Vitamin C inhibited tumour cell growth. It may also have potential anticarcinogenic and chemopreventive activity although the results of these studies are often confl icting (, ).

Conclusion
Testing concentrations of Vitamin C induced diff erent aberrations including the impairment of spindle function. Th e spindle disturbances can result in mitotic arrest, multipolar spindles and multipolar segregation, errors in chromosome segregation, formation of chromosome bridges and chromosome laggards. Th e most frequent irregularities were found in anaphase and telophase. Th e ascorbic acid was not clastogenic except at the highest concentration ( μg/ml) in colchicine-treated cultures, and pulverization of chromosome was observed in colchicine-untreated cultures indicating that certain higher concentrations of ascorbic acid have clastogenic eff ects. A certain number of lymphocytes were arrested at anaphase or telophase (in colchicine-untreated cultures of human lymphocytes) indicating that ascorbic acid may also have inhibited tumour cell growth and have potential anticarcinogenic activity.