Cytotoxicity Induced by Tetracyclines Via Protein Photooxidation
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Hindawi Publishing Corporation Advances in Toxicology Volume 2015, Article ID 787129, 10 pages http://dx.doi.org/10.1155/2015/787129 Research Article Cytotoxicity Induced by Tetracyclines via Protein Photooxidation Domenico Fuoco Drug Discovery Unit, McGill Nutrition and Performance Laboratory, McGill University, 5252 Maisonneuve Street, Montreal, QC,CanadaH4A3S5 Correspondence should be addressed to Domenico Fuoco; [email protected] Received 18 July 2014; Revised 22 February 2015; Accepted 3 March 2015 Academic Editor: Mugimane Manjanatha Copyright © 2015 Domenico Fuoco. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Bacterial ribosomes have been considered the principal targets of tetracyclines. Recently, new clinical data has shown how other biomacromolecules are involved in the cellular damage of bacteria. Researchers are now reconsidering the pharmacological classification of tetracyclines, not only based on their semisynthetic or synthetic generations but also following the new mechanisms of action that are progressively being discovered. Materials and Methods. The toxicity properties of seven tetracycline derivatives (tetracycline, oxytetracycline, demeclocycline, chlortetracycline, doxycycline, minocycline, and meclocycline) were investigated in vitro using a cell line of human keratinocytes. Cells were irradiated in the presence of tetracyclines for different durations and at three different intensities of light. The investigation of protein oxidation was set up using model proteins to quantify the formation of carbonyl groups. Results. After incubation and irradiation with UV light, the viability of keratinocytes was assessed with half the maximal inhibitory concentration for doxycycline, demeclocycline, chlortetracycline, and tetracycline. No phototoxicity was observed for oxytetracycline, meclocycline, and minocycline. Conclusions. This study provides evidence that tetracycline’s derivatives show different photobehaviour according to their chemical properties due to different reactive groups on the same molecular skeleton. 1. Introduction inevitably, cellular death [5–9]. TCs have shown profound activity against mammalian mitochondria [10, 11]. The effect Tetracyclines (TCs) are some of the oldest antibiotics still of TCs on eukaryotic cell membranes is less famous, and used today. Their safety is well assessed and WHO (World it was thought that the selectivity of TCs as antibiotics was Health Organization) considers them integral members of due to their inability to cross mammalian cellular mem- the essential list of medication for underdeveloped countries branes. That notion was dismissed when TCs were found [1]. They are relatively safe and are used to treat various to inhibit the growth of certain mammalian cell lines at infectious diseases [2]. The primary known side effect of TCs concentrations similar to the MIC (Minimum Inhibiting is due to their phototoxicity. They increase skin sensitivity Concentration) values needed to inhibit bacterial growth to light that can lead to significant discoloration (red and [12, 13]. It is well-documented that TCs are able to induce brown spots). Furthermore, the damage can, at times, be photoreactions in human skin and nails [14]. The incidence permanent and lead to more long-term issues, such as skin of skin photosensitivity following treatment with doxycycline cancer [3].TCsareknowntobeassimilatedintheteeth and demeclocycline has been reported to be especially high and bones of young individuals [4].Theaimofthisstudy [6]. To our knowledge, there are no clinical reports of light- is to elucidate the mechanisms of toxicity in mammalian induced side effects from Minocycline. Phototoxicity in vivo cells induced by TCs. It has been postulated that TCs can is partially oxygen dependent and singlet oxygen is possibly inhibit cellular growth in bacteria, specifically, by binding involved [9]. One of the well-studied mechanisms for toxicity to the 30S ribosomal subunit. TCs also change the bacterial in mammalian cells is caused by DNA cleavage due to membrane integrity and mechanical properties, eventually single and double strand breaks induced by complexes of causing macromolecular dysfunction, cellular lysis, and, DNA and photoproducts of TCs [15]. In this study we set 2 Advances in Toxicology Table 1: Molecular structures of the investigated compounds shown were Sigma-Aldrich products used without further purifica- in Scheme 1. tion. Further purification was not required, as these com- Compound Prefix R R R R pounds were purchased with a fluorometric grade [17]. 1 2 3 4 Dimethyl sulfoxide (DMSO) and ethanol (EtOH) were pur- Oxytetracycline Oxy H OH OH OH chased from Fluka and used without further purification. Doxycycline Doxy H CH3 HOH The pH of aqueous solutions was adjusted by Britton buffers Tetracycline Tetra H CH 3 OH H in the pH 2–12 range. Bovine serum albumin (BSA) and Demeclocycline Demeclo Cl H OH H ribonuclease A (RNase A) were purchased from Sigma- Chlortetracycline Chloro Cl CH3 OH H Aldrich (Milano, Italy). Meclocycline Meclo Cl =CH2 —OH Minocycline Mino N(CH3)2 HHH2.2. Cell Strains. Experiments were carried out on an immor- talized, nontumorigenic cell line of human keratinocytes Upper peripheral region (NCTC-2544). The cellular line was grown in Dulbecco’s Modified Eagle Medium (DMEM) medium (Sigma-Aldrich), R4 supplemented with 115 units/mL of penicillin G, 115 g/mL R1 R2 R3 H N(CH3)2 streptomycin, and 10% fetal calf serum (Invitrogen, Milan, OH Italy). The generation time of NCTC-2544 is approximately 21 h. D C B A 2.3. Irradiation Procedure (Light Source). Two HPW 125 CONH2 OH Philips lamps, mainly emitting at 365 nm, were used for OH O OH O irradiation experiments. The spectral irradiance of the source 2 Lower peripheral region was 4.0 mW cm as measured at the sample level by a Cole- Parmer Instrument Company Radiometer (Niles, IL, USA) Scheme 1: Molecular structure of the investigated compounds. equipped with a 365-CX sensor. 2.4. Instruments (Spectrophotometer). Absorption spectra out to determine the role of protein oxidation as another were recorded with a Perkin-Elmer Lambda 800 spectropho- important factor in the phototoxicity mechanism of seven tometer. Fluorescence emission spectra were measured with clinically used TCs (Scheme 1 and Table 1). The rates of a Fluorolog-2 (Spex, F112AI) spectrophotofluorometer. photochemical degradation and the in vitro phototoxicity of the TCs are qualitatively correlated to the clinical pho- 2.5. Photodynamic Inactivation of Cellular Culture (Cellular totoxicity [10]. In agreement with other works referenced Phototoxicity). Phototoxicity experiments were carried out herein, this paper follows the experimental strategy “from on an immortalized, nontumorigenic cell line of human thecelltothebiomolecule.”ThetypicalTCs,thosethatact keratinocytes (NCTC-2544). Cellular line was grown in Dul- as classic protein-synthesis inhibitors, such as tetracycline, becco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich), doxycycline, minocycline, and chlortetracycline, exhibit bac- supplemented with 115 units/mL of penicillin G, 115 ug/mL teriostatic activity, at least initially in bacteria. Other TCs have streptomycin, and 10% fetal calf serum (Invitrogen, Milan, been found to be bactericidal, killing bacteria with an atypical Italy). The generation time of NCTC-2544 is approximately mechanism. Atypical TCs are believed to act by disruption 21 h. Individual wells of a 96-well tissue culture microtiter of cellular membranes, inhibiting all cellular processes and plate (Falcon; Becton-Dickinson) were inoculated with a macromolecular synthesis pathways [16]. Additionally, both complete medium containing NCTC-2544 cells in exponen- ∘ typical and atypical TCs have pharmacological effects against tial growth. The plates were incubated at 37 Cinahumidified eukaryotic cells across multiple cell types; their molecular 5% CO2 incubator for 18 h prior to the experiments. After mechanisms of action are just beginning to be understood. medium removal, a drug solution, previously prepared in A discussion of the effects of TCs against both bacteria and dimethyl sulfoxide and Hank’s balanced salt solution (HBSS, mammalian cells demonstrate the chemically “promiscuous” pH 7.2), was added to each well and the plates were incubated ∘ nature of the tetracycline molecules, as they can interact with at 37 C for 30 min and then irradiated. After irradiation, the a variety of receptors, both prokaryotic and eukaryotic, to solution was replaced with the medium, and the plates were modulate cell processes [16]. incubated for 72 h. After this period, control cells reached a confluence of about 90% and the cell viability was assayed 2. Materials & Methods by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test [18, 19]. 2.1. Chemicals. The investigated compounds belong to the family of TCs (Scheme 1 and Table 1). Tetracycline, oxytetra- 2.6. Protein Oxidation. Solutions of BSA and RNase A cycline dehydrate, demeclocycline hydrochloride, chlortetra- (0.5 mg/mL) in phosphate buffer 10 mM were irradiated cycline hydrochloride, doxycycline hydrochloride, minocy- in the presence of the test compounds for various dura- cline hydrochloride, and meclocycline sulfosalicylate salt tions in a quartz cuvette. At different times, an aliquot of Advances in Toxicology