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Introduction

Tetracyclines are a widely used family of antibiotics (Smilack, 1999). Doxycycline (DOX), which is the most used of the tetracyclines, has bacteriostatic activity against a wide variety of organisms, both Gram-positive and -negative. It is widely used for the treatment of many different bacterial infections and associated conditions, such as Chlamydia, urinary tract, respiratory tract, and gastrointestinal tract infections, acne, gonorrhea, and other diseases (Saikali and Singh, 2003; Jantratid et al., 2010). Because of its high lipid solubility, it has good tis-sue penetration and a prolonged elimination half-life, allowing once-daily dosing (Nguyen et al., 1989; Agwuh and MacGowan, 2006). The bacteriostatic activity of DOX, like all tetracyclines, lies in its capacity to inhibit protein synthesis by preventing the binding of the aminoacyl t-RNA to the ribosome (Clark and Chang, 1965; Saikali and Singh, 2003).

Among a wide variety of pharmaceutical com-pounds, antibiotics are of special concern because of

their extensive use in human and veterinary medicine. Because of the increasing amount in the environ-ment and causing serious problems for human health, biological impacts of antibiotics have been widely discussed (Kolpin et al., 2002; Bautitz and Nogueira, 2007; Çelik and Eke, 2011). It has been shown that some antibiotics also have toxic effects on nontarget organisms (Isidori et al., 2005; Çelik and Eke, 2011). The tetracyclines are closely congeneric derivatives of the polycyclic naphthacene carboxamide (Rawal and Rawal, 2001). Polycyclic aromatic hydrocarbons and heterocyclic aromatic compounds constitute a major class of chemical carcinogens present in the environ-ment. These compounds have the ability to damage the DNA of these compounds and require activation to electrophilic metabolites to exert their mutagenic or carcinogenic effects (Xue and Warshawsky, 2005). DOX is also polycyclic and aromatic in nature (Arshad et al., 2005). Therefore, it is important to determine the genotoxic potential of this drug in humans.

RESEARCH ARTICLE

Genotoxic and cytotoxic effects of doxycycline in cultured human peripheral blood lymphocytes

Zülal Atlı Şekeroğlu, Feridun Afan, and Vedat Şekeroğlu

Faculty of Science and Letters, Department of Biology, Ordu University, Ordu, Turkey

AbstractDoxycycline (DOX) is a broad-spectrum tetracycline antibiotic used in the treatment of many infections. In this study, the genotoxic and cytotoxic effects of DOX in cultured human peripheral blood lymphocytes were investigated by measuring chromosome aberrations (CAs), cytokinesis-block micronucleus (CBMN) assay, mitotic index (MI), and nuclear division index (NDI). Cultures were treated with DOX at three concentrations (2, 4, and 6 µg/mL) for 48 hours. Mitomycin C (MMC) was used as a positive control. All the tested concentrations of DOX for MI and the higher concentrations (4 and 6 µg/mL) for NDI significantly decreased mitotic activity. However, there are no significant differences between negative control and all the tested concentrations of DOX for CA and MN frequencies. In conclusion, our results indicate that DOX has a cytotoxic effect, but not a genotoxic effect, on human peripheral blood lymphocyte cultures. Further detailed studies, especially about the cell-cycle kinetics of DOX, are required to elucidate the decreases in dividing cells and make a possible risk assessment on cells of patients receiving therapy with this drug. Further, if the specific cytostatic and cytotoxic potential of DOX to different types of cancer cells is investigated in detail, it may also have been used as an antitumoral drug.Keywords: Doxycycline, chromosomal aberrations, micronucleus, mitotic index, nuclear division index

Address for Correspondence: Zülal Atlı Şekeroğlu, Faculty of Science and Letters, Department of Biology, Ordu University, 52200 Ordu, Turkey; Fax: +90 452234 44 00; E-mail: zulalas@odu.edu.tr

(Received 04 July 2011; revised 05 August 2011; accepted 10 August 2011)

Drug and Chemical Toxicology, 2012; 35(3): 334–340© 2012 Informa Healthcare USA, Inc.ISSN 0148-0545 print/ISSN 1525-6014 onlineDOI: 10.3109/01480545.2011.621954

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10.3109/01480545.2011.621954

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Worldwide genotoxicity testing of pharmaceuticals before commercialization is mandated by regulatory agencies, because there is a growing concern that some medical products can be mutagenic. From a drug devel-opment standpoint, it is important to have a thorough understanding of the mechanism of any positive genetic toxicity findings so that informed decisions may be made with respect to risk (Snyder and Green, 2001). Because antibiotics have been extensively used in the treatment of many infections, it is necessary to identify their possible genotoxic effects on humans.

Both negative (Kersten et al., 1999) and positive results (Arshad et al., 2005; Dimitrijevic et al., 2006) have been reported regarding the genotoxicity of DOX on vari-ous mammalian cells by using cytogenetic test systems. However, a few genotoxicity studies have been reported in human cells. Taking into account the lack of information, we evaluated the genotoxic and cytotoxic effects of DOX on human peripheral blood lymphocytes using chromo-some aberrations (CAs) and the cytokinesis-block micro-nucleus (CBMN) test and measuring mitotic index (MI) and nuclear division index (NDI), respectively, in vitro.

Methods

ChemicalsDOX (trade names; Adoxa, Atridox, Avidoxy, Doksin, Doryx, Doxoral, Doxyhexal, Doxylin, Microdox, Monodox, Oracea, Oraxyl, Periostat, Tetradox, Vibramycin, and Vibrox) was obtained from Sigma-Aldrich (CAS no.: 24390-14-5; St. Louis, Missouri, USA). The chemical structure of DOX [doxycycline hyclate; ([4S (4aR, 5S, 5aR, 6R, 12aS)] − 4-(dimethylamino) – 1, 4, 4a, 5, 5a, 6, 11, 12a-octahydro-3, 5, 10, 12, 12a-pentahydroxy6 -methyl-1, 11 − deoxonapth-tacene − 2-carboxamide monohydrochloride, compound with ethyl alcohol (2: 1), monohydrate] is shown in Figure 1. The test substance was dissolved in sterile distilled water. Mitomycin-C (MMC) was used as a positive control (Serva, CAS no.: 50-07-7; SERVA Electrophoresis GmbH, Heidelberg, Germany) and was dissolved in sterile distilled water. Cytochalasin-B was obtained from Sigma-Aldrich (CAS no.: 14930-96-2), and colcemid was supplied by Biological Industries (cat. no.: 12-004-1; Kibbutz Beit-Haemek, Israel). Other chemicals used for fixation and stain-ing were obtained from Merck (Darmstadt, Germany).

Donors and collection of blood samplesThis study was carried out by using blood samples from 4 healthy, nonsmoking donors (2 males and 2 females) between 20 and 24 years in age. All volunteers gave

informed consent to participate in the study and signed consent forms. The criteria of acceptability to the experi-ment were having good health, not having serious illness and not receiving any medical therapy, and not using alcohol or drugs or cigarettes. The protocol was approved by the Clinical Research Ethics Committee of the University of Ondokuz (Mayis, Turkey). All blood samples were taken at the same day of the initiation of the experi-ment (between 9.00 a.m. and 9.30 a.m.) to minimize pos-sible confounding effects of dietary factors.

Lymphocyte culturesHeparinized whole blood (0.3 mL) was added to 2.5 mL of RPMI 1640 medium (Biological Industries), supple-mented with 20% fetal bovine serum, 1% antibiotics (penicillin, streptomycin, and amphotericin), 1.2% phytohemagglutinin, and 2% L-glutamine (Biological Industries). In this study, the cytotoxic and genotoxic effects of DOX were tested at 2-, 4-, and 6-μg/mL con-centrations. These concentrations were chosen on the basis of a preliminary cytotoxicity test, an established standard assay for the detection of chemical com-pound genotoxicity on mammalian cell cultures in vitro. According to the cytotoxicity test procedure, we prepared a series of blood lymphocyte cultures of DOX at concentration ranges between 0.5 and 300 μg/mL. MI frequency was scored in each DOX culture as well as in the negative control culture. MI values decreased linearly as DOX concentration increased: 30, 19, 11, 7, and 3 metaphases in 2,000 cells were found at concen-trations of 10, 20, 30, 40, and 50 µg/mL, respectively. No metaphases were observed at concentrations ranging up to 50 µg/mL, and DOX exhibited important cytotoxic effect at these concentrations. A concentration of 6 μg/mL that reduced the MI to approximately 50% (50% inhibitory concentration; IC

50) was used as the highest

dose in the cytogenetic analysis. The concentrations of 4 and 2 μg/mL were used as medium and low doses in this study, respectively. An untreated culture (negative control) and a positive culture treated with MMC (posi-tive control) at a final concentration of 0.16 μg/mL were established, as well.

Experimental results have confirmed that lymphocyte cultures have over half their metaphases in second divi-sion at 48 hours (Poddar at al., 2004). The best period of cultivation of human peripheral lymphocytes for chromosomal analyses in subjects exposed to mutagens and carcinogens is 44–48 hours (Kalina et al., 1990). Therefore, cultures were treated with DOX for 48 hours in this study.

CA assayLymphocyte cultures for CA assay were prepared using the methods developed by Evans (1984). Blood cultures were incubated for 72 hours at 37°C. The test substance, DOX, was added to the cultures at 24 hours. Colcemid, at a final concentration of 0.06 μg/mL, was added to the cultures 2 hours before harvesting. Cells were harvested Figure 1. Chemical structure of doxycycline hyclate.

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by replacing the culture medium with a hypotonic solu-tion (0.4% KCI), in which cells were incubated for 20 min-utes at 37°C. Cells were fixed in a cold Carnoy’s fixative (methanol:glacial acetic acid, 3:1 v/v) three times. To pre-pare the slides, 5 drops of the fixed cell suspension were dropped on clean and cold slides and air-dried. Slides were stained with 5% Giemsa stain solution (diluted with Sorensen buffer, pH 6.8) for 5 minutes.

CBMN assayCBMN assay was carried out using techniques of Fenech (2000), Rothfuss et al. (2000), and Kirsch-Volders et al. (2003). Peripheral lymphocyte cultures were incubated at 37°C for 68 hours and treated with DOX during the last 48 hours. Cytochalasin B (final concentration of 8 μg/mL) was added to arrest cytokinesis at 44 hours after the start of the culture. Cells were harvested by centrifugation, and the pellet was resuspended in hypotonic solution of 0.56% KCI for 5 minutes at 37°C. Cells were fixed in a cold fixative (methanol:glacial ace-tic acid:%0.9 NaCI, 5:1:6 v/v/v). After recentrifugation, cells were refixed two times with methanol:glacial ace-tic acid (5:1 v/v). Slides were prepared by dropping and air-drying. Slides were stained with 5% Giemsa stain solution (diluted with Sorensen buffer, pH 6.8) for 15 minutes.

Slide evaluationSlides were examined using a Leica DM2500 light micro-scope at 1,000× magnification (Leica Microsystems Inc., Buffalo Grove, Illinois, USA). The MI was calculated as the number of metaphases in 2,000 cells analyzed per culture for each treatment and donor. For the analysis of CA, 100 well-spread, intact metaphases were examined per cul-ture for each treatment and donor. The number of each type of aberration, percentages of CA, and aberrant cells with CA were recorded and summarized. Classification of chromosome aberrations was carried out as described in the International System for Cytogenetic Nomenclature (1985). Gaps were not considered as CAs, as recom-mended by Mace et al. (1978).

For the binucleate (BN) cells analysis, the number of BN cells in 2,000 lymphocyte cells was scored per culture. The number of micronuclei in 2,000 BN cells per culture, for each treatment and donor, was scored for the MN analysis.

The criteria to evaluate the BN cells and micronuclei were in accord with the recommendation of Titenko-Holland et al. (1997) and Fenech (2000). For determining NDI, the number of cells with well-preserved cytoplasm, contain-ing 1–4 nuclei in 1,000 cells, was scored per culture for each treatment and donor. The NDI was calculated using the following formula: NDI = 1 × M1 + 2 × M2 + 3 × M3 + 4 × M4/1,000; where M1 through M4 represent the number of cells with 1–4 nuclei (Eastmond and Tucker, 1989; Kocaman and Topaktaş, 2007).

Statistical analysisStatistical analysis was performed using one-way analy-sis of variance. Experimental values are expressed as the mean ± standard error. Comparisons between groups were made using a post-hoc Tukey test. Dose-response relationships were determined from the correlation coefficients (r). P < 0.05 was considered as the level of significance.

Results

Three concentrations (2, 4, and 6 µg/mL) and four cyto-genetic parameters (CA, MN, MI, and NDI) were used to determine the genotoxic and cytotoxic effects of DOX on cultured human peripheral lymphocytes. Table 1 repre-sents the results obtained for CAs and the MI, and Table 2 shows the results obtained for the MN frequencies in BN cells and for the NDI after the treatment of DOX in peripheral blood lymphocyte cultures.

As expected, the positive control (MMC) significantly induced the number of CAs and the frequency of aber-rant cells with CAs (P < 0.05). DOX slightly increased the number of CAs and the frequency of aberrant cells with CAs in a concentration-dependent manner (r = 0.88), but these increases, at all concentrations tested, were statistically not significant, compared with the nega-tive control (P < 0.05). The results of the CBMN assay indicated that DOX slightly induced a concentration- dependent increase in the frequency of BN cells with MN (MNBN) (r = 0.95). But, these increases were statisti-cally not significant, compared to negative control, for MN frequency (P < 0.05).

With respect to the cytotoxic effects of the test com-pound on lymphocyte cultures, as measured by MI

Table 1. Effect of doxycycline (DOX) on mitotic index (MI) and chromosomal aberrations (CA) in human lymphocyte cultures. CA

Treatments MI % ± SE Bʹ Bʹʹ F SCU CE DC P Total CA CA % ± SE Aberrant cells with CA % ± SENegative control 4.03 ± 0.06 8 2 6 1 1 — 1 19 4.75 ± 1.79 4.75 ± 1.79Positive control 0.65 ± 0.10 74 24 92 2 3 2 2 199 49.75 ± 4.32 46.75 ± 3.72

DOX (µg/mL) 2 2.21 ± 0.33* 14 4 10 6 — — 2 36 9.00 ± 0.91 8.50 ± 0.95

4 2.05 ± 0.04* 9 1 16 4 2 — 5 37 9.25 ± 3.42 8.75 ± 3.32

6 1.98 ± 0.23* 23 11 24 5 — — 1 64 16.00 ± 4.41 15.50 ± 4.48

*Significantly different from the negative control; P < 0.05.Bʹ, chromatid breaks; Bʹʹ, chromosome breaks; F, fragments; SCU, sister chromatid unions; CE, chromatid exchanges; DC, dicentric chromosome; P: polyploidy.

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and NDI, a concentration-dependent reduction in cell proliferation was also observed (r = 0.97 and r = 0.95, respectively). All the concentrations of DOX significantly reduced the MI values, compared to negative control (P < 0.05), whereas significant decreases in NDI values were detected at the higher concentrations of DOX (4 and 6 µg/mL), compared to negative control (P < 0.05).

The results of this study indicate that DOX can exert a cytotoxic effect because of statistically significant decreases in MI and NDI, but has no genotoxic activity in both CA and MN assays in human peripheral blood lymphocyte cells.

Discussion

Many positive genetic toxicology test results may arise as a consequence of cytotoxicity, rather than from true drug-DNA interactions. Cytotoxicity may be a result of lysosomal damage and release of DNA endonucleases, adenosine triphosphate ATP depletion, or impairment of mitochondrial function (Galloway, 2000; Snyder and Green, 2001). MI and NDI are used as indicators of ade-quate cell proliferation and cytotoxicity in parallel with other biological endpoints. A decrease in MI and/or NDI reflects an inhibition of cell-cycle progression and/or loss of proliferative capacity. They have also been proven to offer valuable information on the mechanism of action of certain drugs in vitro (Rojas et al., 1993; Anderson et al., 1988; Lopez Nigro et al., 2003; Seligmann et al., 2003). The interaction between cytotoxicity and tumor promotion has been indicated in several studies (Albert and Magee, 2000).

In the present study, DOX has a cytotoxic effect in human blood peripheral lymphocytes as a result of sig-nificantly decreasing the MI and NDI. These decreases could be caused by an inhibition of DNA synthesis or a blocking in the G

2 phase of the cell cycle, which pre-

vents the cell from entering into mitosis. There are sev-eral results showing similar effects as DOX. Tetracycline showed significant decreases in the MI, NDI, and prolif-eration index (PI) at concentrations of 0.1, 0.5, 1, 1.5, and 2 μg/mL in human peripheral lymphocytes in a concen-tration-dependent manner. DOX significantly decreased the PI in the presence of UV in Chinese hamster V79 cells

at concentrations of 0.05, 0.46, 4.62, 46.2, and 462 µg/mL and showed a phototoxic effect (Kersten et al., 1999). It has been reported that tetracyclines also showed an inhi-bition of growth of carcinogen-induced tumors (Kroon et al., 1984) and had cytotoxic and cytostatic effects on various cultured human cancer cells (Saikali and Singh, 2003). Specific cytostatic activity resulting from the selec-tive permeability of different cells to tetracyclines was also tested, and it has been reported that DOX inhibited tumor cell proliferation in permeable T-cell leukemia of the rat (Van den Bogert et al., 1985). DOX was found to be cytostatic to human renal and prostate carcinoma, osteosarcoma, and mesothelioma cells (Van den Bogert et al., 1986a; Fife et al., 1997, 1998; Rubins et al., 2001). Cytostatic activity of tetracyclines was confirmed in fibroblasts and sarcoma cells, and the proliferation arrest in these cells was shown to be caused by an accumula-tion of the cell population in the G

1 phase of the cell cycle

(Van den Bogert et al., 1986b). Because of the similarity between the prokaryotic protein synthesis machinery and that of eukaryotic mitochondria, tetracyclines are also able to interfere with mitochondrial protein syn-thesis in mammalian cells. The selective permeability of different mammalian cells to tetracyclines led to the hypothesis that these agents could be used to achieve cell-proliferation arrest and applied to the treatment of malignancies (Kroon and Van den Bogert, 1983; Saikali and Singh, 2003).

Cytogenetic methods are commonly used in humans to monitor exposure to potential mutagens/carcino-gens, and positive responses should be regarded as indicators of genotoxic damage. The CA assay provides a sensitive biomarker of genotoxicity and detects chromo-somal damage induced after one cell division; structural changes in the chromosomes are evaluated while cells are in the metaphase stage of division (Preston et al., 1987; Karahalil et al., 2005). In mitotically active cells, MN arises from structural chromosomal aberrations or from whole chromosomes, and it was concluded that the monitoring of MN frequency in peripheral blood lymphocytes from human individuals could be a very effective test in the estimation of the effects of bio-logical, physical, and chemical agents (Fenech, 2000; Dimitrijevic et al., 2006).

Table 2. Effect of doxycycline (DOX) on binucleate (BN) cells, micronucleated binucleate (MNBN) cells, and nuclear division index (NDI) in human lymphocyte cultures

Distribution of BN cells according to

no. of micronuclei Distribution of cellsaccording to

no. of nuclei Treatments 0 1 2 3 >3 MNBN % ± SE 1 2 3 4 NDI ± SENegative control 7,990 10 0.12 ± 0.01 684 3,083 117 116 1.91 ± 0.02Positive control 7,521 433 33 11 2 5.98 ± 0.31 2,911 1,054 21 14 1.28 ± 0.07DOX (µg/mL) 2 7,987 14 — — — 0.17 ± 0.01 922 2,968 54 56 1.80 ± 0.01 4 7,984 16 — — — 0.20 ± 0.02 1,295 2,696 5 4 1.67 ± 0.01* 6 7,976 23 1 — — 0.30 ± 0.07 1,485 2,496 13 6 1.63 ± 0.02**Significantly different from the negative control; P < 0.05.SE, standard error.

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DNA damage caused through the reactive metabo-lites of polycyclic aromatic hydrocarbons and hetero-cyclic aromatic compounds are described involving the DNA covalent binding to form stable or depurinating adducts, the formation of apurinic sites, and oxidative damage (Xue and Warshawsky, 2005). Tetracyclines are a group of broad-spectrum antibiotics (Smilack, 1999), and their structure is described as derivatives of the polycyclic napthalene carboxamide (Rawal and Rawal, 2001). In various cells, some in vitro and in vivo genetic toxicology tests, to have the cytotoxical and genotoxical information about the risk of some tetracycline antibiot-ics, have given different results. Several studies have con-cluded that tetracycline has no genotoxic effect (Zeiger and Haworth, 1985; Suzuki, 1987; Anderson et al., 1990; Hagiwara et al., 2006), whereas some researchers found positive (Tsutsui et al., 1976; Çelik and Eke, 2011) and equivocal results (Myhr et al., 1990). Oxytetracycline was found to be genotoxic and mutagenic in several studies (Blitek et al., 1983; Myhr et al., 1990; McGregor et al., 1991), whereas many reports indicated that it has no genotoxic and mutagenic effects (Andrews et al., 1980; Mortelmans et al., 1986; Anderson et al., 1990; Kersten et al., 1999; Isidori et al., 2005). According to the unpub-lished reports of the U.S. Food and Drug Administration (FDA) and the Physicians’ Desk Reference (PDR), DOX showed genotoxic effect in mouse lymphoma assay (con-centration was not explained) (FDA, 2004). Doses of 500, 800, and 1,250 mg/kg of DOX did not induce the MN fre-quency in polychromatic erythrocytes in mice, whereas it showed a weak clastogenic effect in cultured Chinese hamster ovary (CHO) cells at concentrations ranging up to 350 µg/mL (FDA, 1997; PDR, 2005). Our results indicated that DOX slightly increased the frequencies of CA and MN in a concentration-dependent manner, but these increases were not significantly different from the negative control culture in human peripheral blood lymphocyte cells under in vitro conditions. According to these results, it seems that human cells are more sensi-tive to DOX than CHO cells. DOX did not enhance the UV-induced MN frequency in Chinese hamster V79 cells at concentrations of 0.05, 0.46, 4.62, 46.2, and 462 µg/mL (Kersten et al., 1999). Percutaneous doses of 40, 80, and 160 mg/kg of DOX induced DNA damage in blood and five mice organs (liver, kidney, lungs, spleen, and heart) in a dose-dependent manner in the alkaline single-cell gel electrophoresis (Comet) assay and showed acute genotoxic effect at higher doses (Arshad et al., 2005). The frequency of MN was measured before and after DOX therapy in the lymphocytes of 38 newly diagnosed adult women with genital Chlamydia trachomatis infec-tion. In the patients who received oral DOX during 10 days (2 × 100 mg per day) and then for another 10 days (1 × 100 mg per day), the MN frequency after the end of therapy was significantly higher than before treatment (Dimitrijevic et al., 2006). The differences between the results of previous results and those of the current study regarding the genotoxicity of DOX may result from the

differences of concentration and treatment time of DOX and cells and/or organisms used.

It has been stated that tetracycline is a bifunctional alkylating agent that probably needs high concentrations to induce CAs (Lawley and Brookes, 1963). Because no metaphases were observed at concentrations ranging up to 50 µg/mL in our preliminary study, cytogenetic analy-sis was not performed in cells exposed to these doses of DOX. This evidence seems to support the hypothesis that this drug causes significant mitotic inhibition and DNA damage resulting from the serious cytotoxic effect and shows clastogenic effect at higher concentrations, as reported by FDA (1997) and PDR (2005).

Conclusions

The results obtained in this study showed the ability of DOX to induce cytotoxicity decreasing the MI and NDI; mean-while, no genotoxic effects were observed using CA and MN assays in human blood lymphocytes under the experimen-tal conditions. Further, human blood lymphocytes were found to be more sensitive to DOX, even at low concentra-tions. These results raise the question about the safety of this drug. Therefore, this antibiotic should be closely monitored to reduce the cytoxic effect, reveal the genetic interactions, develop a comprehensive picture of clinical safety, and bal-ance efficacy with minimizing of cytotoxic and genotoxic effects. Our results may introduce new information open for alternative interpretations. Further detailed cytogenetic studies, especially about the cell-cycle kinetics of DOX, are required to elucidate the decreases in dividing cells in dif-ferent cells and make a possible risk assessment on cells of patients receiving therapy with this drug. Further, if the specific cytostatic and cytotoxic potential of DOX to differ-ent types of cancer cells is investigated in detail, it may also be used as an antitumoral drug, in addition to its antimi-crobial activity, in medical applications.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

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