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Cytotoxicity Induced by Tetracyclines via Protein Photooxidation

Cytotoxicity Induced by Tetracyclines via Protein Photooxidation Cytotoxicity Induced by Tetracyclines via Protein Photooxidation div.banner_title_bkg div.trangle { border-color: #376240 transparent transparent transparent; opacity:0.8; /*new styles start*/ -ms-filter:"progid:DXImageTransform.Microsoft.Alpha(Opacity=80)" ;filter: alpha(opacity=80); /*new styles end*/ } div.banner_title_bkg_if div.trangle { border-color: transparent transparent #376240 transparent ; opacity:0.8; /*new styles start*/ -ms-filter:"progid:DXImageTransform.Microsoft.Alpha(Opacity=80)" ;filter: alpha(opacity=80); /*new styles end*/ } div.banner_title_bkg div.trangle { width: 221px; } #banner { background-image: url('http://images.hindawi.com/journals/atox/atox.banner.jpg'); background-position: 50% 0;} Hindawi Publishing Corporation Home Journals About Us Advances in Toxicology About this Journal Submit a Manuscript Table of Contents Journal Menu About this Journal · Abstracting and Indexing · Advance Access · Aims and Scope · Article Processing Charges · Articles in Press · Author Guidelines · Bibliographic Information · Contact Information · Editorial Board · Editorial Workflow · Free eTOC Alerts · Publication Ethics · Reviewers Acknowledgment · Submit a Manuscript · Subscription Information · Table of Contents Open Special Issues · Special Issue Guidelines Abstract Full-Text PDF Full-Text HTML Full-Text ePUB Linked References How to Cite this Article Advances in Toxicology Volume 2015 (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, Canada H4A 3S5 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. Abstract 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 Tetracyclines (TCs) are some of the oldest antibiotics still used today. Their safety is well assessed and WHO (World Health Organization) considers them integral members of the essential list of medication for underdeveloped countries [ 1 ]. They are relatively safe and are used to treat various infectious diseases [ 2 ]. The primary known side effect of TCs is due to their phototoxicity. They increase skin sensitivity to light that can lead to significant discoloration (red and brown spots). Furthermore, the damage can, at times, be permanent and lead to more long-term issues, such as skin cancer [ 3 ]. TCs are known to be assimilated in the teeth and bones of young individuals [ 4 ]. The aim of this study is to elucidate the mechanisms of toxicity in mammalian cells induced by TCs. It has been postulated that TCs can inhibit cellular growth in bacteria, specifically, by binding to the 30S ribosomal subunit. TCs also change the bacterial membrane integrity and mechanical properties, eventually causing macromolecular dysfunction, cellular lysis, and, inevitably, cellular death [ 5 – 9 ]. TCs have shown profound activity against mammalian mitochondria [ 10 , 11 ]. The effect of TCs on eukaryotic cell membranes is less famous, and it was thought that the selectivity of TCs as antibiotics was due to their inability to cross mammalian cellular membranes. That notion was dismissed when TCs were found to inhibit the growth of certain mammalian cell lines at concentrations similar to the MIC (Minimum Inhibiting Concentration) values needed to inhibit bacterial growth [ 12 , 13 ]. It is well-documented that TCs are able to induce photoreactions in human skin and nails [ 14 ]. The incidence of skin photosensitivity following treatment with doxycycline and demeclocycline has been reported to be especially high [ 6 ]. To our knowledge, there are no clinical reports of light-induced side effects from Minocycline. Phototoxicity in vivo is partially oxygen dependent and singlet oxygen is possibly involved [ 9 ]. One of the well-studied mechanisms for toxicity in mammalian cells is caused by DNA cleavage due to single and double strand breaks induced by complexes of DNA and photoproducts of TCs [ 15 ]. In this study we set out to determine the role of protein oxidation as another important factor in the phototoxicity mechanism of seven clinically used TCs (Scheme 1 and Table 1 ). The rates of photochemical degradation and the in vitro phototoxicity of the TCs are qualitatively correlated to the clinical phototoxicity [ 10 ]. In agreement with other works referenced herein, this paper follows the experimental strategy “from the cell to the biomolecule.” The typical TCs, those that act as classic protein-synthesis inhibitors, such as tetracycline, doxycycline, minocycline, and chlortetracycline, exhibit bacteriostatic activity, at least initially in bacteria. Other TCs have been found to be bactericidal, killing bacteria with an atypical mechanism. Atypical TCs are believed to act by disruption of cellular membranes, inhibiting all cellular processes and macromolecular synthesis pathways [ 16 ]. Additionally, both typical and atypical TCs have pharmacological effects against eukaryotic cells across multiple cell types; their molecular mechanisms of action are just beginning to be understood. A discussion of the effects of TCs against both bacteria and mammalian cells demonstrate the chemically “promiscuous” nature of the tetracycline molecules, as they can interact with a variety of receptors, both prokaryotic and eukaryotic, to modulate cell processes [ 16 ]. Table 1: Molecular structures of the investigated compounds shown in Scheme 1 . Scheme 1: Molecular structure of the investigated compounds. 2. Materials & Methods 2.1. Chemicals The investigated compounds belong to the family of TCs (Scheme 1 and Table 1 ). Tetracycline, oxytetracycline dehydrate, demeclocycline hydrochloride, chlortetracycline hydrochloride, doxycycline hydrochloride, minocycline hydrochloride, and meclocycline sulfosalicylate salt were Sigma-Aldrich products used without further purification. Further purification was not required, as these compounds were purchased with a fluorometric grade [ 17 ]. Dimethyl sulfoxide (DMSO) and ethanol (EtOH) were purchased from Fluka and used without further purification. The pH of aqueous solutions was adjusted by Britton buffers in the pH 2–12 range. Bovine serum albumin (BSA) and ribonuclease A (RNase A) were purchased from Sigma-Aldrich (Milano, Italy). 2.2. Cell Strains Experiments were carried out on an immortalized, nontumorigenic cell line of human keratinocytes (NCTC-2544). The cellular line was grown in Dulbecco’s Modified Eagle Medium (DMEM) medium (Sigma-Aldrich), supplemented with 115 units/mL of penicillin G, 115 μ g/mL streptomycin, and 10% fetal calf serum (Invitrogen, Milan, Italy). The generation time of NCTC-2544 is approximately 21 h. 2.3. Irradiation Procedure (Light Source) Two HPW 125 Philips lamps, mainly emitting at 365 nm, were used for irradiation experiments. The spectral irradiance of the source was 4.0 mW cm 2 as measured at the sample level by a Cole-Parmer Instrument Company Radiometer (Niles, IL, USA) equipped with a 365-CX sensor. 2.4. Instruments (Spectrophotometer) Absorption spectra were recorded with a Perkin-Elmer Lambda 800 spectrophotometer. Fluorescence emission spectra were measured with a Fluorolog-2 (Spex, F112AI) spectrophotofluorometer. 2.5. Photodynamic Inactivation of Cellular Culture (Cellular Phototoxicity) Phototoxicity experiments were carried out on an immortalized, nontumorigenic cell line of human keratinocytes (NCTC-2544). Cellular line was grown in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich), supplemented with 115 units/mL of penicillin G, 115 ug/mL streptomycin, and 10% fetal calf serum (Invitrogen, Milan, Italy). The generation time of NCTC-2544 is approximately 21 h. Individual wells of a 96-well tissue culture microtiter plate (Falcon; Becton-Dickinson) were inoculated with a complete medium containing NCTC-2544 cells in exponential growth. The plates were incubated at 37°C in a humidified 5% CO 2 incubator for 18 h prior to the experiments. After medium removal, a drug solution, previously prepared in dimethyl sulfoxide and Hank’s balanced salt solution (HBSS, pH 7.2), was added to each well and the plates were incubated at 37°C for 30 min and then irradiated. After irradiation, the solution was replaced with the medium, and the plates were incubated for 72 h. After this period, control cells reached a confluence of about 90% and the cell viability was assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test [ 18 , 19 ]. 2.6. Protein Oxidation Solutions of BSA and RNase A (0.5 mg/mL) in phosphate buffer 10 mM were irradiated in the presence of the test compounds for various durations in a quartz cuvette. At different times, an aliquot of the solution was taken and the degree of protein oxidation was monitored spectrophotometrically, by derivatization with 2,4-dinitrophenylhydrazine (DNPH) [ 20 ]. Fluorescence was measured with Fluorolog-2 (Spex, F112AI) spectrophotofluorometer, 330 nm and at 392 nm for BSA and with 274 nm and at 303 nm for RNase A. The rational for protein study via fluorescence quenching is very well-known since the work of Eftink of 1981 [ 21 ]. 2.7. Statistical Data Analysis Unless indicated differently, the results are presented as mean ± SEM. The differences between irradiated and nonirradiated sample were analyzed using the two-sided Student’s -test. 2.8. Computational Data Analysis and Method The partition coefficient ( ) organic phase/aqueous phase was determined using semiempirical software with a computational method according to Ghose-Crippen [ 22 , 23 ]. The values of were then used to calculate the distribution coefficients ( ) using a commercial software (Marvin, Chemaxon Company) for physical chemistry prediction. is an important parameter to understand the distribution of drugs in the human body under physiological conditions. The collected data is summarized in Table 2 and is very important to better understand the behaviour of TCs in vivo [ 14 , 15 ]. Table 2: and are data available from the database of http://pubchem.ncbi.nlm.nih.gov according to the Ghose-Crippen method [ 22 , 23 ]. Serum protein binding and renal clearance data are available from the database of http://www.drugbank.ca/ . 3. Results Our approach for elucidating the molecular mechanism of TCs induced phototoxicity was to estimate whether or not a correlation existed between relative clinical phototoxicity of a series of TCs and in vitro assay (Scheme 2 ). The four assays chosen were (a) the relative rates of TCs photodegradation, (b) the relative phototoxicity of TCs to human keratinocytes, (c) the binding to model proteins (BSA and RNase A), and (d) the photoreaction with the proteins and the relative amount of photooxidation. Scheme 2: Strategy of experiment. The four assays chosen were (a) the relative rates of TCs photodegradation, (b) the relative phototoxicity of TCs to human keratinocytes, (c) the binding to model proteins (BSA and RNase A), and (d) the photoreaction with specific amino acids and the relative amount of photo oxidation. 3.1. Role of Lipophilicity The aim of this research was to clarify which TCs were phototoxic and which did not show phototoxicity and to understand the different mechanisms involved in cellular damage. TCs fall within a very similar range of molecular weights, between 444 and 500 Da. However, they differ greatly in their partition coefficients ( ), as well as distribution coefficients ( ), which range from negative to positive values [ 16 , 17 , 24 ]. The degree of lipophilicity was in accordance with the data found in current literature [ 22 ]: tetracycline > meclocycline > oxytetracycline (Figure 1 ). Only data related to photocytotoxicity will be demonstrated and discussed in the following paragraphs. However, in a future paper, currently in preparation, many other physical-chemical measures will be analyzed, namely, in an elaborate table. Figure 1: Fluorometric titration of some TCs upon different concentration of BSA. emission with BSA (bound molecule) and emission without BSA (free molecule). The graph shows that the affinity between the molecules and BSA increases with lipophilicity: tetracycline > meclocycline > oxytetracycline. Data expressed in this graph are in accord with those reported in Table 2 . 3.2. Cytotoxicity and Photocytotoxicity None of the compounds being investigated have shown a cytotoxic effect in the absence of irradiation. Our results confirm the toxicity of tetracycline, chlortetracycline, doxycycline, and demeclocycline. The relative scale of toxicity magnification ranges from the most to the least toxic (at the maximum irradiation time): doxycycline > chlortetracycline > demeclocycline > tetracycline. Oxytetracycline, meclocycline, and minocycline do not show phototoxicity (Figure 2 ). The results are in agreement with the clinical data published for these molecules [ 6 ] and with other in vitro experiments performed on different cellular lines [ 25 , 26 ]. Specifically, it is possible to observe a trend of toxicity that is similar to the lipophilicity degree, as shown in Table 2 . Minocycline is the least phototoxic in vitro and clinically. It is not reported to be a photosensitizer (Figure 2 ). Doxycycline and chlortetracycline, followed by demeclocycline and tetracycline, are the strongest photosensitizers when tested on normal human keratinocytes. Figure 2: MTT test of photocytotoxicity of seven tetracycline derivatives. Significance: therapeutic agents with an IC 50 under 10 μ M are considered extremely cytotoxic; range between 50 and 100 μ M is considered moderately cytotoxic. All TCs studied in this work have noncytotoxic effect on keratinocytes with no light exposure but have toxicity effect in the presence of light. If toxicity is shown upper 200 μ M, the compounds are considered safe for their therapeutic purpose. 3.3. Model Protein Binding Two spectroscopic techniques, fluorescence and fluorescence-quenching, were used to evaluate the binding of TCs with BSA. The fluorescence spectrum of the molecules upon addition of the protein was monitored. In this case, a change in the fluorescence spectrum should be observed for the changed conditions of the bound molecule (Figure 1 ). The graph in Figure 1 shows that the affinity between the molecules and BSA increases with lipophilicity: tetracycline > meclocycline > oxytetracycline. That rate of affinity shown in Figure 1 is in accordance with the literature data [ 27 – 31 ]. 3.4. Photoreaction on Model Proteins (BSA and RNase A) When looking at demeclocycline with BSA, a reduction in the fluorescence peak of 70% at a dose of 15 J/cm 2 of UVA was observed. Contrastingly, with the RNase, a 23% reduction is evident at the same experimental conditions (UVA dose and drug concentration) (Figure 3 ). For doxycycline and minocycline with BSA, a reduction in the fluorescence peak of 55% at a dose of 15 J/cm 2 of UVA was observed, whereas, with the RNase, a 19% reduction is evident at the same experimental conditions (UVA dose and drug concentration) (Figure 3 ). The differences in the photo-behaviour of the proteins can be attributed to the different amino acids on the structure of the proteins (more units of tryptophan in BSA and more units of tyrosine in RNase). Figure 3: Photoreaction of some tetracyclines with same concentration of model proteins at different time of irradiation. On the right, photoreaction upon BSA and on the left upon RNase A. emission with protein (bound molecules) and emission without protein (free molecule). 3.5. Determination of the Formation of Carbonyls in Two Model Proteins The production of carbonyls, and thus the photooxidation induced by demeclocycline, is greater than that caused by doxycycline and minocycline (Figures 4 – 6 ). In fact, this difference can be attributed to the activity of photoproducts, especially for doxycycline [ 32 , 33 ]. Photoreaction in the relationship between the concentration of TCs and induced damage varies from 70% to 20% of maximum fluorescence emission. Literature reports that tryptophan is the most susceptible residue to photooxidation on BSA, while tyrosine is the main photo-damaged residue in RNase A. Results, herein presented, are in perfect accord with the data available in literature [ 33 – 35 ]. Figure 4: Formation of carbonyl of model proteins photo-induced alone (control) and then with different concentrations of demeclocycline. On the right is oxidation effect on BSA and on the left is the same reaction on RNase A. Figure 5: Formation of carbonyl of model proteins photo-induced alone (control) and then with different concentrations of doxycycline. On the right is the oxidation effect on BSA and on the left is the same reaction on RNase A. Figure 6: Formation of carbonyl of model proteins photo-induced alone (control) and then with different concentration of minocycline. On the right is the oxidation effect on BSA and on the left is the same reaction on RNase A. 3.6. Photodegradation by UVA and UVB Irradiation It is well known that TCs, when irradiated in solution, photodegrade [ 36 – 45 ]. Particularly in aqueous solution, the most stable of our research compounds is minocycline. However, the medium for cytotoxicity assay is in DMSO and water (50 : 50). Hence, a new trend for degradation was studied in organic medium (Figure 7 ). To get a preliminary indication of the photolysis, compounds were studied for their absorption spectra after irradiation with increasing doses of UVA (365 nm) and UVB (319 nm). The absorbance after exposure to UV radiation is given by the sum of absorbances of all species present in the solution (Figure 7 ). Figure 8 shows the kinetics of first-order in which the molecules are photodegraded under UVA condition. The intensity of damage is almost equal for both UVA and UVB (only kinetics plot for UVA are shown in the present paper). Our results suggest that the observed trend of photodegradation is indicative of a scale of toxicity. Under those experimental conditions, we list from the most to the least stable: tetracycline > demeclocycline > chlortetracycline > meclocycline > doxycycline. Except for meclocycline, all the TCs just mentioned are those responsible for the phototoxicity effect on human keratinocytes. Figure 7: Photodegradation of minocycline dissolved in a mixture of phosphate buffer and DMSO (50 : 50). Graphs show, on the left, exposure at UVA and, on the right, at UVB. Figure 8: Photodegradation of all tetracyclines dissolved in a mixture of phosphate buffer and DMSO (50 : 50). The graph shows the kinetics of first-order in which the molecules are photodegraded ( value of absorbance after the solution has absorbed 15 J/cm 2 of irradiation in 30 minutes value of absorbance at 0 J/cm 2 ). 4. Discussion Despite having the same rigid skeleton, which is founded on four condensate rings, TCs show differences in their physical-chemistry and biological-chemistry [ 16 ]. Each TC presents specific chemical groups on the own skeleton, which strongly affect their lipophilicity (Table 2 ). Those differences in lipophilicity, among the TCs, can explain their reactivity with different biological targets. The lipophilicity also affects oral absorption and the ability to penetrate the blood-brain barrier; only minocycline and doxycycline cross it to a measurable extent [ 46 ]. As TCs become more lipophilic, they also become more serum protein bound [ 28 ]. This changes their overall bioavailability, maximum detectable concentration, and tetracycline half-life [ 47 ]. According to this hypothesis, the role of lipophilicity, pharmacokinetics data was collected to improve our knowledge of the binding characteristics of serum albumin for TCs [ 28 , 48 ]. These findings, about the biochemistry of TCs, are in agreement with the results of Ljunggren [ 49 ]. The correlation of our results, with previous in vitro studies using lymphocytes [ 50 ] and erythrocytes [ 51 , 52 ], is fairly good. This experimental data is in agreement with clinical reports and comparative phototoxicity trials in humans [ 6 ]. The incidence of photosensitivity reactions to the tetracycline antibiotics varies with the structure of the drug: demeclocycline > tetracycline > minocycline (Figure 2 ). Chlortetracyclines (demeclocycline and chlortetracycline), the most rapidly photolyzed compounds, are less phototoxic than doxycycline, which is the most potent photosensitizer of the TCs. Because the TCs photodegraded during the phototoxicity assay, it is possible that the photodegradation product contributed to the phototoxicity. Photodegradation is dependent on several factors: concentration of dissolved oxygen, reaction conditions, type of buffer solution, time of irradiation, and presence of antioxidant agents [ 42 ]. Meclocycline is a special case since it is the only chlortetracycline derivative without phototoxic effect. In fact, there are no clinical reports available about its phototoxicity. Our approach was to describe the molecular mechanism of TCs induced phototoxicity in order to estimate whether or not a correlation existed between relative clinical phototoxicity of a series of TCs and an in vitro assay. The four assays chosen were (a) the relative rates of TCs photodegradation, (b) the relative phototoxicity of TCs to human keratinocytes, (c) the binding to model proteins (BSA and RNase A), and (d) the photoreaction with the proteins and the relative amount of photooxidation. The clinical photosensitizing ability of all the compounds used in this study was estimated from scattered reports in the literature. It is clear that the members of the TC family most frequently reported to cause photosensitivity are the chlortetracyclines (demeclocycline and chlortetracycline). In trial studies, demeclocycline and chlortetracycline-induced photosensitivity were observed in 90%–100% of the subjects and doxycycline-induced photosensitivity in about 20% [ 6 ]. The variation of in vivo photosensitizing ability agrees with the trend of TCs in relative photodegradation rates that were observed (Figure 8 ). Indeed, it is possible to deduce that photoproducts contribute significantly to the phototoxic process. In fact, the basis for the reported differences between the in vivo action and the in vitro behaviour may be in the different structures of relative photoproducts. The contribution of photoproducts of TCs to the mechanism of toxicity has been well known since the 80s [ 28 ] and, today, it is even proposed in therapy, as reported by Jiao et al. [ 53 ]. To better understand all the factors involved in the mechanism of action of TCs, their interaction with proteins was assessed. Proteins represent 68% of the dry weight of cells and tissues and, therefore, may be accessible targets for different photosensitizers. Photooxidation can induce many changes in a protein: fragmentation, aggregation, oxidation of amino acids, denaturation, changes in its proteolytic susceptibility, alteration of the surface hydrophobicity, and changes in its structure. In this work, the photosensitizing effects of tetracyclines were studied mainly on two models of proteins: bovine serum albumin (BSA, Mw > 60.000 Da, a transport protein, essential to maintaining the osmotic gradient in cell) and bovine ribonuclease A (RNase A, Mw > 14.000 Da, an enzyme capable of hydrolysing the phosphodiester bonds of RNA). 5. Conclusion and Future Perspective Tetracyclines used at therapeutic concentrations do not have toxicity in mammalian cells, but after UVA/UVB exposure, they show phototoxicity (doxycycline, demeclocycline, chlortetracycline, and tetracycline). This behaviour is caused by their photodegradation products and their reactive nature. The mechanism of cellular damage is associated with an increase of oxidation in biomacromolecules such as albumin (BSA) and RNase A. Not all the tetracyclines, once irradiated, result in phototoxicity. Minocycline is the most photostable compound of this series and does not show phototoxicity. All compounds share the same molecular skeleton composed of four condensed aromatic rings. However, the different reactive groups produce different chemical properties and, in the end, different biological activities. It was determined that tetracyclines have ability to form a new structure with the proteins without irradiation. It was also observed that their affinity for albumin increases with the lipophilicity of tetracyclines. Subsequently, their interaction with proteins following irradiation was studied and then correlated with clinical data. The results of this study will be helpful for all laboratories that are currently developing the next generation of tetracyclines, in order to have the maximum efficacy and fewer side effects. The experiments confirm the literature data and introduce new information about the mechanisms of toxicity in keratinocytes, which proves to be of essential utility to clinical treatments. Executive Summary Photobehaviour of Tetracyclines under UVA and UVB Lights . (i) Absorption spectrum of tetracyclines shows different rates of degradation and the same trend under UVA and UVB conditions. These differences are associated to the reactivity of different chemical groups upon the same molecular skeleton. (ii) Photoaffinity of studied compounds for albumin and others proteins is related to the different lipophilic proprieties shown for the seven tetracyclines derivatives. Phototoxicity of Seven Tetracyclines Derivatives . (i) Incubation of human keratinocytes for 30 min with decreasing concentration of seven compounds followed by irradiation with blue light (6.25 J/cm 2 ) was necessary to measure IC 50 . (ii) Doxycycline is the most phototoxic compound of the series, followed by demeclocycline, chlortetracycline, and tetracycline. Minocycline is the more photostable compound and has no phototoxicity as well as oxytetracycline and meclocycline. Protein Oxidation as Primary Target of the Mechanism of Toxicity . (i) Carbonyl assay was performed with two model proteins (BSA and RNase A) to study the entity and quantify the cellular damage via oxidation of specific aromatic amino acids. Conflict of Interests Author declares no conflict of interests. Acknowledgments Some instruments and facilities used in this paper were offered from the Laboratories of Photobiology (Department of Pharmaceutical Science, University of Padua) and Photochemistry (Department of Chemistry, University of Perugia) during the author’s doctoral thesis. Grateful thanks goes to Professor Francesco Dall’Acqua and Professor Fausto Elisei for supervising this research project. The author wishes to thank Karina Mastronardi for her comments in the editing of this paper. References World Health Organization, 8th WHO Model List of Essential Medicines , WHO Press, Geneva, Switzerland, 2013. I. Chopra and M. Roberts, “Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance,” Microbiology and Molecular Biology Reviews , vol. 65, no. 2, pp. 232–260, 2001. View at Publisher · View at Google Scholar · View at Scopus T. P. Habif, “Images in clinical medicine. Doxycycline-induced phototoxicity,” The New England Journal of Medicine , vol. 355, no. 2, p. 182, 2006. View at Publisher · View at Google Scholar · View at Scopus A. R. Sánchez, R. S. Rogers III, and P. J. Sheridan, “Tetracycline and other tetracycline-derivative staining of the teeth and oral cavity,” International Journal of Dermatology , vol. 43, no. 10, pp. 709–715, 2004. View at Publisher · View at Google Scholar · View at Scopus M. L. Nelson, “Chemical and biological dynamics of tetracyclines,” Advances in Dental Research , vol. 12, no. 2, pp. 5–11, 1998. View at Publisher · View at Google Scholar · View at Scopus M. L. Nelson and M. Y. Ismail, “The antibiotic and nonantibiotic tetracyclines,” in Comprehensive Medical Chemistry II , pp. 598–617, Elsevier, Amsterdam, The Netherlands, 2007. View at Google Scholar S. W. Anson, C. F. Chingnell, P. Roethling, and B. K. Cummings, “O 2 -photogenerated from aqueous solution of tetracycline antibiotics (pH 7.3) as evidenced by DMPO spin trapping and cytochrome C reduction,” Biochemical and Biophysical Research Communications , vol. 146, no. 3, pp. 1191–1195, 1987. View at Google Scholar A. S. Li, C. F. Chignell, and R. D. Hall, “Cutaneous phototoxicity of tetracycline antibiotics: generation of free radicals and singlet oxygen during photolysis as measured by spin-trapping and the phosphorescence of singlet molecular oxygen,” Photochemistry and Photobiology , vol. 46, no. 3, pp. 379–382, 1987. View at Publisher · View at Google Scholar · View at Scopus J. P. Martin, K. Colina, and N. Logsdon, “Role oxygen radicals in the phototoxicity of tetracyclines toward Escherichia coli B,” Journal of Bacteriology , vol. 169, no. 6, pp. 2516–2522, 1987. View at Google Scholar · View at Scopus T. Hasan, I. E. Kochevar, D. J. McAuliffe, B. S. Cooperman, and D. Abdulah, “Mechanism of tetracycline phototoxicity,” The Journal of Investigative Dermatology , vol. 83, no. 3, pp. 179–183, 1984. View at Publisher · View at Google Scholar · View at Scopus E. Ahler, W. J. Sullivan, A. Cass et al., “Doxycycline aAlters metabolism and proliferation of human cell line,” PLoS ONE , vol. 8, no. 5, Article ID e64561, 2013. View at Publisher · View at Google Scholar · View at Scopus A. Sigler, P. Schubert, W. Hillen, and M. Niederweis, “Permeation of tetracyclines through membranes of liposomes and Escherichia coli ,” European Journal of Biochemistry , vol. 267, no. 2, pp. 527–534, 2000. View at Publisher · View at Google Scholar · View at Scopus L. Brunton, B. Chabner, and B. Knollman, Goodman and Gilman's the Pharmacological Basis of Therapeutics , McGraw-Hill, New York, NY, USA, 10th edition, 2001. M. Nelson, W. Hillen, and R. A. Greenwald, Tetracyclines in Biology, Chemistry and Medicine , Birkhäuser, Basel, Switzerland, 2001. M. A. Khan, J. Mustafa, and J. Musarrat, “Mechanism of DNA strand breakage induced by photosensitized tetracycline-Cu(II) complex,” Mutation Research: Fundamental and Molecular Mechanisms of Mutagenesis , vol. 525, no. 1-2, pp. 109–119, 2003. View at Publisher · View at Google Scholar · View at Scopus D. Fuoco, “Classification framework and chemical biology of tetracycline-structure-based drugs,” Antibiotics , vol. 1, no. 1, pp. 1–13, 2012. View at Publisher · View at Google Scholar B. Carlotti, D. Fuoco, and F. Elisei, “Fast and ultrafast spectroscopic investigation of tetracycline derivatives in organic and aqueous media,” Physical Chemistry Chemical Physics , vol. 12, no. 48, pp. 15580–15591, 2010. View at Publisher · View at Google Scholar · View at Scopus N. J. Traynor, M. D. Barratt, W. W. Lovell, J. Ferguson, and N. K. Gibbs, “Comparison of an in vitro cellular phototoxicity model against controlled clinical trials of fluoroquinolone skin phototoxicity,” Toxicology in Vitro , vol. 14, no. 3, pp. 275–283, 2000. View at Publisher · View at Google Scholar · View at Scopus H. Spielmann, W. V. Lovell, and E. Holzle, “In vitro phototoxicity testing,” ATLA , vol. 22, pp. 314–348, 1994. View at Google Scholar J. M. Fagan, B. G. Sleczka, and I. Sohar, “Quantitation of oxidative damage to tissue proteins,” The International Journal of Biochemistry & Cell Biology , vol. 31, no. 7, pp. 751–757, 1999. View at Publisher · View at Google Scholar · View at Scopus M. R. Eftink and C. A. Ghiron, “Fluorescence quenching studies with proteins,” Analytical Biochemistry , vol. 114, no. 2, pp. 199–227, 1981. View at Publisher · View at Google Scholar · View at Scopus H. Terada, “Proposed partition mechanism of tetracycline,” Chemical and Pharmaceutical Bulletin , vol. 9, pp. 1965–1975, 1977. View at Google Scholar · View at Scopus A. K. Ghose and G. M. Crippen, “Atomic physicochemical parameters for three-dimensional-structure-directed quantitative structure-activity relationships. 2. Modeling dispersive and hydrophobic interactions,” Journal of Chemical Information and Computer Science , vol. 27, pp. 21–35, 1987. View at Google Scholar · View at Scopus F. Bahrami, D. L. Morris, and M. H. Pourgholami, “Tetracyclines: drugs with huge therapeutic potential,” Mini-Reviews in Medicinal Chemistry , vol. 12, no. 1, pp. 44–52, 2012. View at Publisher · View at Google Scholar · View at Scopus M. Bjellerup, T. Kjellstrom, and B. Ljunggren, “Influence of tetracycline phototoxicity on the growth of cultured human fibroblasts,” Journal of Investigative Dermatology , vol. 85, no. 6, pp. 573–574, 1985. View at Publisher · View at Google Scholar · View at Scopus A. M. Drucker and C. F. Rosen, “Drug-induced photosensitivity: culprit drugs, management and prevention,” Drug Safety , vol. 34, no. 10, pp. 821–837, 2011. View at Publisher · View at Google Scholar · View at Scopus E. L. Gelamo and M. Tabak, “Spectroscopic studies on the interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants,” Spectrochimica Acta—Part A: bolecular and Biomolecular Spectroscopy , vol. 56, no. 11, pp. 2255–2271, 2000. View at Publisher · View at Google Scholar · View at Scopus M. A. Khan, S. Muzammil, and J. Musarrat, “Differential binding of tetracyclines with serum albumin and induced structural alterations in drug-bound protein,” International Journal of Biological Macromolecules , vol. 30, no. 5, pp. 243–249, 2002. View at Publisher · View at Google Scholar · View at Scopus C. Jiang and L. Luo, “Spectrofluorimetric determination of human serum albumin using a doxycycline-europium probe,” Analytica Chimica Acta , vol. 506, no. 2, pp. 171–175, 2004. View at Publisher · View at Google Scholar · View at Scopus S. Bi, D. Song, Y. Tian, X. Zhou, Z. Liu, and H. Zhang, “Molecular spectroscopic study on the interaction of tetracyclines with serum albumins,” Spectrochimica Acta Part A , vol. 61, pp. 629–636, 2005. View at Publisher · View at Google Scholar M. I. Burgos, R. A. Fernández, M. S. Celej, L. I. Rossi, G. D. Fidelio, and S. A. Dassie, “Binding of the highly toxic tetracycline derivative, anhydrotetracycline, to bovine serum albumin,” Biological and Pharmaceutical Bulletin , vol. 34, no. 8, pp. 1301–1306, 2011. View at Publisher · View at Google Scholar · View at Scopus A. M. Layton and W. J. Cunliffe, “Phototoxic eruptions due to doxycycline—a dose-related phenomenon,” Clinical and Experimental Dermatology , vol. 18, no. 5, pp. 425–427, 1993. View at Publisher · View at Google Scholar · View at Scopus C. R. Shea, G. A. Olack, H. Morrison, N. Chen, and T. Hasan, “Phototoxicity of lumidoxycycline,” Journal of Investigative Dermatology , vol. 101, no. 3, pp. 329–333, 1993. View at Publisher · View at Google Scholar · View at Scopus A. Catalfo, G. Bracchitta, and G. de Guidi, “Role of aromatic amino acid tryptophan UVA-photoproducts in the determination of drug photosensitization mechanism: a comparison between methylene blue and naproxen,” Photochemical and Photobiological Sciences , vol. 8, no. 10, pp. 1467–1475, 2009. View at Publisher · View at Google Scholar · View at Scopus Q. H. Song, H. B. Wang, W. J. Tang, Q. X. Guo, and S. Q. Yu, “Model studies of the (6–4) photoproduct photoreactivation: efficient photosensitized splitting of thymine oxetane units by covalently linked tryptophan in high polarity solvents,” Organic and Biomolecular Chemistry , vol. 4, no. 2, pp. 291–298, 2006. View at Publisher · View at Google Scholar · View at Scopus J. A. M. Wiebe and D. E. Moore, “Oxidation photosensitized by tetracyclines,” Journal of Pharmaceutical Sciences , vol. 66, no. 2, pp. 186–189, 1977. View at Publisher · View at Google Scholar · View at Scopus H. Oka, Y. Ikai, N. Kawamura et al., “Photodecomposition products of tetracycline in aqueous solution,” Journal of Agricultural and Food Chemistry , vol. 37, no. 1, pp. 226–231, 1989. View at Publisher · View at Google Scholar · View at Scopus R. E. Drexel, G. Olack, C. Jones, G. N. Chmurny, R. Santini, and H. Morrison, “Lumitetracycline: a novel new tetracycline photoproduct,” Journal of Organic Chemistry , vol. 55, no. 8, pp. 2471–2478, 1990. View at Publisher · View at Google Scholar · View at Scopus G. Olack and H. Morrison, “Formation and characterization of lumitetracycline-type photoproducts from members of the tetracycline family,” Journal of Organic Chemistry , vol. 56, no. 16, pp. 4969–4971, 1991. View at Publisher · View at Google Scholar · View at Scopus M. M. Beliakova, S. I. Bessonov, B. M. Sergeyev, I. G. Smirnova, E. N. Dobrov, and A. M. Kopylov, “Rate of tetracycline photolysis during irradiation at 365 nm,” Biochemistry , vol. 68, no. 2, pp. 182–187, 2003. View at Publisher · View at Google Scholar · View at Scopus M. Addamo, V. Augugliaro, A. di Paola et al., “Removal of drugs in aqueous systems by photoassisted degradation,” Journal of Applied Electrochemistry , vol. 35, no. 7-8, pp. 765–774, 2005. View at Publisher · View at Google Scholar · View at Scopus J. J. López-Peñalver, M. Sánchez-Polo, C. V. Gómez-Pacheco, and J. Rivera-Utrilla, “Photodegradation of tetracyclines in aqueous solution by using UV and UV/H 2 O 2 oxidation processes,” Journal of Chemical Technology and Biotechnology , vol. 85, no. 10, pp. 1325–1333, 2010. View at Publisher · View at Google Scholar · View at Scopus A. Pena, L. P. Palilis, C. M. Lino, M. I. Silveira, and A. C. Calokerinos, “Determination of tetracycline and its major degradation products by chemiluminescence,” Analytica Chimica Acta , vol. 405, no. 1-2, pp. 51–56, 2000. View at Publisher · View at Google Scholar · View at Scopus Y. Chen, C. Hu, J. Qu, and M. Yang, “Photodegradation of tetracycline and formation of reactive oxygen species in aqueous tetracycline solution under simulated sunlight irradiation,” Journal of Photochemistry and Photobiology A: Chemistry , vol. 197, no. 1, pp. 81–87, 2008. View at Publisher · View at Google Scholar · View at Scopus A. Davies, G. O. Phillips, and A. G. Reid, “Photochemical oxidation of tetracycline in aqueous solution,” Journal of Chemical Spectroscopy Perkin II , pp. 369–375, 1979. View at Google Scholar · View at Scopus C. Chryssanthou, B. Graber, S. Mendelson, and G. Goldstein, “Increased blood-brain barrier permeability to tetracycline in rabbits under dysbaric conditions,” Undersea Biomedical Research , vol. 6, no. 4, pp. 319–328, 1979. View at Google Scholar · View at Scopus T. Rodgers and M. Rowland, “Physiologically based pharmacokinetic modelling 2: predicting the tissue distribution of acids, very weak bases, neutrals and zwitterions,” Journal of Pharmaceutical Sciences , vol. 95, no. 6, pp. 1238–1257, 2006. View at Publisher · View at Google Scholar · View at Scopus M. S. von Wittenau, “Some pharmacokinetic aspects of doxycycline metabolism in man.,” Chemotherapy , vol. 13, pp. 41–50, 1968. View at Publisher · View at Google Scholar · View at Scopus B. Ljunggren and M. Bjellerup, “Double blind cross-over studies on phototoxicity to three tetracycline derivatives in human volunteers,” Photodermatology , vol. 4, no. 6, pp. 281–287, 1987. View at Google Scholar · View at Scopus S. Sandberg, J. Glette, G. Hopen, and C. O. Solberg, “Doxycycline induced photodamage to Human Neutrophils and tryptophan,” Photochemistry and Photobiology , vol. 39, no. 1, pp. 43–48, 1984. View at Publisher · View at Google Scholar · View at Scopus R. Nilsson, G. Swanbeck, and G. Wennersten, “Primary mechanisms of erythrocyte photolysis induced by biological sensitizers and phototoxic drugs,” Photochemistry and Photobiology , vol. 22, no. 5, pp. 183–186, 1975. View at Publisher · View at Google Scholar · View at Scopus M. Bjellerup and B. Ljunggren, “Photohemolytic potency of tetracyclines,” Journal of Investigative Dermatology , vol. 84, no. 4, pp. 262–264, 1985. View at Publisher · View at Google Scholar · View at Scopus S. Jiao, S. Zheng, D. Yin, L. Wang, and L. Chen, “Aqueous photolysis of tetracycline and toxicity of photolytic products to luminescent bacteria,” Chemosphere , vol. 73, no. 3, pp. 377–382, 2008. 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Cytotoxicity Induced by Tetracyclines via Protein Photooxidation

Advances in Toxicology , Volume 2015 (2015) – Mar 24, 2015

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Cytotoxicity Induced by Tetracyclines via Protein Photooxidation div.banner_title_bkg div.trangle { border-color: #376240 transparent transparent transparent; opacity:0.8; /*new styles start*/ -ms-filter:"progid:DXImageTransform.Microsoft.Alpha(Opacity=80)" ;filter: alpha(opacity=80); /*new styles end*/ } div.banner_title_bkg_if div.trangle { border-color: transparent transparent #376240 transparent ; opacity:0.8; /*new styles start*/ -ms-filter:"progid:DXImageTransform.Microsoft.Alpha(Opacity=80)" ;filter: alpha(opacity=80); /*new styles end*/ } div.banner_title_bkg div.trangle { width: 221px; } #banner { background-image: url('http://images.hindawi.com/journals/atox/atox.banner.jpg'); background-position: 50% 0;} Hindawi Publishing Corporation Home Journals About Us Advances in Toxicology About this Journal Submit a Manuscript Table of Contents Journal Menu About this Journal · Abstracting and Indexing · Advance Access · Aims and Scope · Article Processing Charges · Articles in Press · Author Guidelines · Bibliographic Information · Contact Information · Editorial Board · Editorial Workflow · Free eTOC Alerts · Publication Ethics · Reviewers Acknowledgment · Submit a Manuscript · Subscription Information · Table of Contents Open Special Issues · Special Issue Guidelines Abstract Full-Text PDF Full-Text HTML Full-Text ePUB Linked References How to Cite this Article Advances in Toxicology Volume 2015 (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, Canada H4A 3S5 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. Abstract 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 Tetracyclines (TCs) are some of the oldest antibiotics still used today. Their safety is well assessed and WHO (World Health Organization) considers them integral members of the essential list of medication for underdeveloped countries [ 1 ]. They are relatively safe and are used to treat various infectious diseases [ 2 ]. The primary known side effect of TCs is due to their phototoxicity. They increase skin sensitivity to light that can lead to significant discoloration (red and brown spots). Furthermore, the damage can, at times, be permanent and lead to more long-term issues, such as skin cancer [ 3 ]. TCs are known to be assimilated in the teeth and bones of young individuals [ 4 ]. The aim of this study is to elucidate the mechanisms of toxicity in mammalian cells induced by TCs. It has been postulated that TCs can inhibit cellular growth in bacteria, specifically, by binding to the 30S ribosomal subunit. TCs also change the bacterial membrane integrity and mechanical properties, eventually causing macromolecular dysfunction, cellular lysis, and, inevitably, cellular death [ 5 – 9 ]. TCs have shown profound activity against mammalian mitochondria [ 10 , 11 ]. The effect of TCs on eukaryotic cell membranes is less famous, and it was thought that the selectivity of TCs as antibiotics was due to their inability to cross mammalian cellular membranes. That notion was dismissed when TCs were found to inhibit the growth of certain mammalian cell lines at concentrations similar to the MIC (Minimum Inhibiting Concentration) values needed to inhibit bacterial growth [ 12 , 13 ]. It is well-documented that TCs are able to induce photoreactions in human skin and nails [ 14 ]. The incidence of skin photosensitivity following treatment with doxycycline and demeclocycline has been reported to be especially high [ 6 ]. To our knowledge, there are no clinical reports of light-induced side effects from Minocycline. Phototoxicity in vivo is partially oxygen dependent and singlet oxygen is possibly involved [ 9 ]. One of the well-studied mechanisms for toxicity in mammalian cells is caused by DNA cleavage due to single and double strand breaks induced by complexes of DNA and photoproducts of TCs [ 15 ]. In this study we set out to determine the role of protein oxidation as another important factor in the phototoxicity mechanism of seven clinically used TCs (Scheme 1 and Table 1 ). The rates of photochemical degradation and the in vitro phototoxicity of the TCs are qualitatively correlated to the clinical phototoxicity [ 10 ]. In agreement with other works referenced herein, this paper follows the experimental strategy “from the cell to the biomolecule.” The typical TCs, those that act as classic protein-synthesis inhibitors, such as tetracycline, doxycycline, minocycline, and chlortetracycline, exhibit bacteriostatic activity, at least initially in bacteria. Other TCs have been found to be bactericidal, killing bacteria with an atypical mechanism. Atypical TCs are believed to act by disruption of cellular membranes, inhibiting all cellular processes and macromolecular synthesis pathways [ 16 ]. Additionally, both typical and atypical TCs have pharmacological effects against eukaryotic cells across multiple cell types; their molecular mechanisms of action are just beginning to be understood. A discussion of the effects of TCs against both bacteria and mammalian cells demonstrate the chemically “promiscuous” nature of the tetracycline molecules, as they can interact with a variety of receptors, both prokaryotic and eukaryotic, to modulate cell processes [ 16 ]. Table 1: Molecular structures of the investigated compounds shown in Scheme 1 . Scheme 1: Molecular structure of the investigated compounds. 2. Materials & Methods 2.1. Chemicals The investigated compounds belong to the family of TCs (Scheme 1 and Table 1 ). Tetracycline, oxytetracycline dehydrate, demeclocycline hydrochloride, chlortetracycline hydrochloride, doxycycline hydrochloride, minocycline hydrochloride, and meclocycline sulfosalicylate salt were Sigma-Aldrich products used without further purification. Further purification was not required, as these compounds were purchased with a fluorometric grade [ 17 ]. Dimethyl sulfoxide (DMSO) and ethanol (EtOH) were purchased from Fluka and used without further purification. The pH of aqueous solutions was adjusted by Britton buffers in the pH 2–12 range. Bovine serum albumin (BSA) and ribonuclease A (RNase A) were purchased from Sigma-Aldrich (Milano, Italy). 2.2. Cell Strains Experiments were carried out on an immortalized, nontumorigenic cell line of human keratinocytes (NCTC-2544). The cellular line was grown in Dulbecco’s Modified Eagle Medium (DMEM) medium (Sigma-Aldrich), supplemented with 115 units/mL of penicillin G, 115 μ g/mL streptomycin, and 10% fetal calf serum (Invitrogen, Milan, Italy). The generation time of NCTC-2544 is approximately 21 h. 2.3. Irradiation Procedure (Light Source) Two HPW 125 Philips lamps, mainly emitting at 365 nm, were used for irradiation experiments. The spectral irradiance of the source was 4.0 mW cm 2 as measured at the sample level by a Cole-Parmer Instrument Company Radiometer (Niles, IL, USA) equipped with a 365-CX sensor. 2.4. Instruments (Spectrophotometer) Absorption spectra were recorded with a Perkin-Elmer Lambda 800 spectrophotometer. Fluorescence emission spectra were measured with a Fluorolog-2 (Spex, F112AI) spectrophotofluorometer. 2.5. Photodynamic Inactivation of Cellular Culture (Cellular Phototoxicity) Phototoxicity experiments were carried out on an immortalized, nontumorigenic cell line of human keratinocytes (NCTC-2544). Cellular line was grown in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich), supplemented with 115 units/mL of penicillin G, 115 ug/mL streptomycin, and 10% fetal calf serum (Invitrogen, Milan, Italy). The generation time of NCTC-2544 is approximately 21 h. Individual wells of a 96-well tissue culture microtiter plate (Falcon; Becton-Dickinson) were inoculated with a complete medium containing NCTC-2544 cells in exponential growth. The plates were incubated at 37°C in a humidified 5% CO 2 incubator for 18 h prior to the experiments. After medium removal, a drug solution, previously prepared in dimethyl sulfoxide and Hank’s balanced salt solution (HBSS, pH 7.2), was added to each well and the plates were incubated at 37°C for 30 min and then irradiated. After irradiation, the solution was replaced with the medium, and the plates were incubated for 72 h. After this period, control cells reached a confluence of about 90% and the cell viability was assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test [ 18 , 19 ]. 2.6. Protein Oxidation Solutions of BSA and RNase A (0.5 mg/mL) in phosphate buffer 10 mM were irradiated in the presence of the test compounds for various durations in a quartz cuvette. At different times, an aliquot of the solution was taken and the degree of protein oxidation was monitored spectrophotometrically, by derivatization with 2,4-dinitrophenylhydrazine (DNPH) [ 20 ]. Fluorescence was measured with Fluorolog-2 (Spex, F112AI) spectrophotofluorometer, 330 nm and at 392 nm for BSA and with 274 nm and at 303 nm for RNase A. The rational for protein study via fluorescence quenching is very well-known since the work of Eftink of 1981 [ 21 ]. 2.7. Statistical Data Analysis Unless indicated differently, the results are presented as mean ± SEM. The differences between irradiated and nonirradiated sample were analyzed using the two-sided Student’s -test. 2.8. Computational Data Analysis and Method The partition coefficient ( ) organic phase/aqueous phase was determined using semiempirical software with a computational method according to Ghose-Crippen [ 22 , 23 ]. The values of were then used to calculate the distribution coefficients ( ) using a commercial software (Marvin, Chemaxon Company) for physical chemistry prediction. is an important parameter to understand the distribution of drugs in the human body under physiological conditions. The collected data is summarized in Table 2 and is very important to better understand the behaviour of TCs in vivo [ 14 , 15 ]. Table 2: and are data available from the database of http://pubchem.ncbi.nlm.nih.gov according to the Ghose-Crippen method [ 22 , 23 ]. Serum protein binding and renal clearance data are available from the database of http://www.drugbank.ca/ . 3. Results Our approach for elucidating the molecular mechanism of TCs induced phototoxicity was to estimate whether or not a correlation existed between relative clinical phototoxicity of a series of TCs and in vitro assay (Scheme 2 ). The four assays chosen were (a) the relative rates of TCs photodegradation, (b) the relative phototoxicity of TCs to human keratinocytes, (c) the binding to model proteins (BSA and RNase A), and (d) the photoreaction with the proteins and the relative amount of photooxidation. Scheme 2: Strategy of experiment. The four assays chosen were (a) the relative rates of TCs photodegradation, (b) the relative phototoxicity of TCs to human keratinocytes, (c) the binding to model proteins (BSA and RNase A), and (d) the photoreaction with specific amino acids and the relative amount of photo oxidation. 3.1. Role of Lipophilicity The aim of this research was to clarify which TCs were phototoxic and which did not show phototoxicity and to understand the different mechanisms involved in cellular damage. TCs fall within a very similar range of molecular weights, between 444 and 500 Da. However, they differ greatly in their partition coefficients ( ), as well as distribution coefficients ( ), which range from negative to positive values [ 16 , 17 , 24 ]. The degree of lipophilicity was in accordance with the data found in current literature [ 22 ]: tetracycline > meclocycline > oxytetracycline (Figure 1 ). Only data related to photocytotoxicity will be demonstrated and discussed in the following paragraphs. However, in a future paper, currently in preparation, many other physical-chemical measures will be analyzed, namely, in an elaborate table. Figure 1: Fluorometric titration of some TCs upon different concentration of BSA. emission with BSA (bound molecule) and emission without BSA (free molecule). The graph shows that the affinity between the molecules and BSA increases with lipophilicity: tetracycline > meclocycline > oxytetracycline. Data expressed in this graph are in accord with those reported in Table 2 . 3.2. Cytotoxicity and Photocytotoxicity None of the compounds being investigated have shown a cytotoxic effect in the absence of irradiation. Our results confirm the toxicity of tetracycline, chlortetracycline, doxycycline, and demeclocycline. The relative scale of toxicity magnification ranges from the most to the least toxic (at the maximum irradiation time): doxycycline > chlortetracycline > demeclocycline > tetracycline. Oxytetracycline, meclocycline, and minocycline do not show phototoxicity (Figure 2 ). The results are in agreement with the clinical data published for these molecules [ 6 ] and with other in vitro experiments performed on different cellular lines [ 25 , 26 ]. Specifically, it is possible to observe a trend of toxicity that is similar to the lipophilicity degree, as shown in Table 2 . Minocycline is the least phototoxic in vitro and clinically. It is not reported to be a photosensitizer (Figure 2 ). Doxycycline and chlortetracycline, followed by demeclocycline and tetracycline, are the strongest photosensitizers when tested on normal human keratinocytes. Figure 2: MTT test of photocytotoxicity of seven tetracycline derivatives. Significance: therapeutic agents with an IC 50 under 10 μ M are considered extremely cytotoxic; range between 50 and 100 μ M is considered moderately cytotoxic. All TCs studied in this work have noncytotoxic effect on keratinocytes with no light exposure but have toxicity effect in the presence of light. If toxicity is shown upper 200 μ M, the compounds are considered safe for their therapeutic purpose. 3.3. Model Protein Binding Two spectroscopic techniques, fluorescence and fluorescence-quenching, were used to evaluate the binding of TCs with BSA. The fluorescence spectrum of the molecules upon addition of the protein was monitored. In this case, a change in the fluorescence spectrum should be observed for the changed conditions of the bound molecule (Figure 1 ). The graph in Figure 1 shows that the affinity between the molecules and BSA increases with lipophilicity: tetracycline > meclocycline > oxytetracycline. That rate of affinity shown in Figure 1 is in accordance with the literature data [ 27 – 31 ]. 3.4. Photoreaction on Model Proteins (BSA and RNase A) When looking at demeclocycline with BSA, a reduction in the fluorescence peak of 70% at a dose of 15 J/cm 2 of UVA was observed. Contrastingly, with the RNase, a 23% reduction is evident at the same experimental conditions (UVA dose and drug concentration) (Figure 3 ). For doxycycline and minocycline with BSA, a reduction in the fluorescence peak of 55% at a dose of 15 J/cm 2 of UVA was observed, whereas, with the RNase, a 19% reduction is evident at the same experimental conditions (UVA dose and drug concentration) (Figure 3 ). The differences in the photo-behaviour of the proteins can be attributed to the different amino acids on the structure of the proteins (more units of tryptophan in BSA and more units of tyrosine in RNase). Figure 3: Photoreaction of some tetracyclines with same concentration of model proteins at different time of irradiation. On the right, photoreaction upon BSA and on the left upon RNase A. emission with protein (bound molecules) and emission without protein (free molecule). 3.5. Determination of the Formation of Carbonyls in Two Model Proteins The production of carbonyls, and thus the photooxidation induced by demeclocycline, is greater than that caused by doxycycline and minocycline (Figures 4 – 6 ). In fact, this difference can be attributed to the activity of photoproducts, especially for doxycycline [ 32 , 33 ]. Photoreaction in the relationship between the concentration of TCs and induced damage varies from 70% to 20% of maximum fluorescence emission. Literature reports that tryptophan is the most susceptible residue to photooxidation on BSA, while tyrosine is the main photo-damaged residue in RNase A. Results, herein presented, are in perfect accord with the data available in literature [ 33 – 35 ]. Figure 4: Formation of carbonyl of model proteins photo-induced alone (control) and then with different concentrations of demeclocycline. On the right is oxidation effect on BSA and on the left is the same reaction on RNase A. Figure 5: Formation of carbonyl of model proteins photo-induced alone (control) and then with different concentrations of doxycycline. On the right is the oxidation effect on BSA and on the left is the same reaction on RNase A. Figure 6: Formation of carbonyl of model proteins photo-induced alone (control) and then with different concentration of minocycline. On the right is the oxidation effect on BSA and on the left is the same reaction on RNase A. 3.6. Photodegradation by UVA and UVB Irradiation It is well known that TCs, when irradiated in solution, photodegrade [ 36 – 45 ]. Particularly in aqueous solution, the most stable of our research compounds is minocycline. However, the medium for cytotoxicity assay is in DMSO and water (50 : 50). Hence, a new trend for degradation was studied in organic medium (Figure 7 ). To get a preliminary indication of the photolysis, compounds were studied for their absorption spectra after irradiation with increasing doses of UVA (365 nm) and UVB (319 nm). The absorbance after exposure to UV radiation is given by the sum of absorbances of all species present in the solution (Figure 7 ). Figure 8 shows the kinetics of first-order in which the molecules are photodegraded under UVA condition. The intensity of damage is almost equal for both UVA and UVB (only kinetics plot for UVA are shown in the present paper). Our results suggest that the observed trend of photodegradation is indicative of a scale of toxicity. Under those experimental conditions, we list from the most to the least stable: tetracycline > demeclocycline > chlortetracycline > meclocycline > doxycycline. Except for meclocycline, all the TCs just mentioned are those responsible for the phototoxicity effect on human keratinocytes. Figure 7: Photodegradation of minocycline dissolved in a mixture of phosphate buffer and DMSO (50 : 50). Graphs show, on the left, exposure at UVA and, on the right, at UVB. Figure 8: Photodegradation of all tetracyclines dissolved in a mixture of phosphate buffer and DMSO (50 : 50). The graph shows the kinetics of first-order in which the molecules are photodegraded ( value of absorbance after the solution has absorbed 15 J/cm 2 of irradiation in 30 minutes value of absorbance at 0 J/cm 2 ). 4. Discussion Despite having the same rigid skeleton, which is founded on four condensate rings, TCs show differences in their physical-chemistry and biological-chemistry [ 16 ]. Each TC presents specific chemical groups on the own skeleton, which strongly affect their lipophilicity (Table 2 ). Those differences in lipophilicity, among the TCs, can explain their reactivity with different biological targets. The lipophilicity also affects oral absorption and the ability to penetrate the blood-brain barrier; only minocycline and doxycycline cross it to a measurable extent [ 46 ]. As TCs become more lipophilic, they also become more serum protein bound [ 28 ]. This changes their overall bioavailability, maximum detectable concentration, and tetracycline half-life [ 47 ]. According to this hypothesis, the role of lipophilicity, pharmacokinetics data was collected to improve our knowledge of the binding characteristics of serum albumin for TCs [ 28 , 48 ]. These findings, about the biochemistry of TCs, are in agreement with the results of Ljunggren [ 49 ]. The correlation of our results, with previous in vitro studies using lymphocytes [ 50 ] and erythrocytes [ 51 , 52 ], is fairly good. This experimental data is in agreement with clinical reports and comparative phototoxicity trials in humans [ 6 ]. The incidence of photosensitivity reactions to the tetracycline antibiotics varies with the structure of the drug: demeclocycline > tetracycline > minocycline (Figure 2 ). Chlortetracyclines (demeclocycline and chlortetracycline), the most rapidly photolyzed compounds, are less phototoxic than doxycycline, which is the most potent photosensitizer of the TCs. Because the TCs photodegraded during the phototoxicity assay, it is possible that the photodegradation product contributed to the phototoxicity. Photodegradation is dependent on several factors: concentration of dissolved oxygen, reaction conditions, type of buffer solution, time of irradiation, and presence of antioxidant agents [ 42 ]. Meclocycline is a special case since it is the only chlortetracycline derivative without phototoxic effect. In fact, there are no clinical reports available about its phototoxicity. Our approach was to describe the molecular mechanism of TCs induced phototoxicity in order to estimate whether or not a correlation existed between relative clinical phototoxicity of a series of TCs and an in vitro assay. The four assays chosen were (a) the relative rates of TCs photodegradation, (b) the relative phototoxicity of TCs to human keratinocytes, (c) the binding to model proteins (BSA and RNase A), and (d) the photoreaction with the proteins and the relative amount of photooxidation. The clinical photosensitizing ability of all the compounds used in this study was estimated from scattered reports in the literature. It is clear that the members of the TC family most frequently reported to cause photosensitivity are the chlortetracyclines (demeclocycline and chlortetracycline). In trial studies, demeclocycline and chlortetracycline-induced photosensitivity were observed in 90%–100% of the subjects and doxycycline-induced photosensitivity in about 20% [ 6 ]. The variation of in vivo photosensitizing ability agrees with the trend of TCs in relative photodegradation rates that were observed (Figure 8 ). Indeed, it is possible to deduce that photoproducts contribute significantly to the phototoxic process. In fact, the basis for the reported differences between the in vivo action and the in vitro behaviour may be in the different structures of relative photoproducts. The contribution of photoproducts of TCs to the mechanism of toxicity has been well known since the 80s [ 28 ] and, today, it is even proposed in therapy, as reported by Jiao et al. [ 53 ]. To better understand all the factors involved in the mechanism of action of TCs, their interaction with proteins was assessed. Proteins represent 68% of the dry weight of cells and tissues and, therefore, may be accessible targets for different photosensitizers. Photooxidation can induce many changes in a protein: fragmentation, aggregation, oxidation of amino acids, denaturation, changes in its proteolytic susceptibility, alteration of the surface hydrophobicity, and changes in its structure. In this work, the photosensitizing effects of tetracyclines were studied mainly on two models of proteins: bovine serum albumin (BSA, Mw > 60.000 Da, a transport protein, essential to maintaining the osmotic gradient in cell) and bovine ribonuclease A (RNase A, Mw > 14.000 Da, an enzyme capable of hydrolysing the phosphodiester bonds of RNA). 5. Conclusion and Future Perspective Tetracyclines used at therapeutic concentrations do not have toxicity in mammalian cells, but after UVA/UVB exposure, they show phototoxicity (doxycycline, demeclocycline, chlortetracycline, and tetracycline). This behaviour is caused by their photodegradation products and their reactive nature. The mechanism of cellular damage is associated with an increase of oxidation in biomacromolecules such as albumin (BSA) and RNase A. Not all the tetracyclines, once irradiated, result in phototoxicity. Minocycline is the most photostable compound of this series and does not show phototoxicity. All compounds share the same molecular skeleton composed of four condensed aromatic rings. However, the different reactive groups produce different chemical properties and, in the end, different biological activities. It was determined that tetracyclines have ability to form a new structure with the proteins without irradiation. It was also observed that their affinity for albumin increases with the lipophilicity of tetracyclines. Subsequently, their interaction with proteins following irradiation was studied and then correlated with clinical data. The results of this study will be helpful for all laboratories that are currently developing the next generation of tetracyclines, in order to have the maximum efficacy and fewer side effects. The experiments confirm the literature data and introduce new information about the mechanisms of toxicity in keratinocytes, which proves to be of essential utility to clinical treatments. Executive Summary Photobehaviour of Tetracyclines under UVA and UVB Lights . (i) Absorption spectrum of tetracyclines shows different rates of degradation and the same trend under UVA and UVB conditions. These differences are associated to the reactivity of different chemical groups upon the same molecular skeleton. (ii) Photoaffinity of studied compounds for albumin and others proteins is related to the different lipophilic proprieties shown for the seven tetracyclines derivatives. Phototoxicity of Seven Tetracyclines Derivatives . (i) Incubation of human keratinocytes for 30 min with decreasing concentration of seven compounds followed by irradiation with blue light (6.25 J/cm 2 ) was necessary to measure IC 50 . (ii) Doxycycline is the most phototoxic compound of the series, followed by demeclocycline, chlortetracycline, and tetracycline. Minocycline is the more photostable compound and has no phototoxicity as well as oxytetracycline and meclocycline. Protein Oxidation as Primary Target of the Mechanism of Toxicity . (i) Carbonyl assay was performed with two model proteins (BSA and RNase A) to study the entity and quantify the cellular damage via oxidation of specific aromatic amino acids. Conflict of Interests Author declares no conflict of interests. Acknowledgments Some instruments and facilities used in this paper were offered from the Laboratories of Photobiology (Department of Pharmaceutical Science, University of Padua) and Photochemistry (Department of Chemistry, University of Perugia) during the author’s doctoral thesis. Grateful thanks goes to Professor Francesco Dall’Acqua and Professor Fausto Elisei for supervising this research project. The author wishes to thank Karina Mastronardi for her comments in the editing of this paper. References World Health Organization, 8th WHO Model List of Essential Medicines , WHO Press, Geneva, Switzerland, 2013. I. Chopra and M. Roberts, “Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance,” Microbiology and Molecular Biology Reviews , vol. 65, no. 2, pp. 232–260, 2001. View at Publisher · View at Google Scholar · View at Scopus T. P. Habif, “Images in clinical medicine. Doxycycline-induced phototoxicity,” The New England Journal of Medicine , vol. 355, no. 2, p. 182, 2006. View at Publisher · View at Google Scholar · View at Scopus A. R. Sánchez, R. S. Rogers III, and P. J. Sheridan, “Tetracycline and other tetracycline-derivative staining of the teeth and oral cavity,” International Journal of Dermatology , vol. 43, no. 10, pp. 709–715, 2004. View at Publisher · View at Google Scholar · View at Scopus M. L. Nelson, “Chemical and biological dynamics of tetracyclines,” Advances in Dental Research , vol. 12, no. 2, pp. 5–11, 1998. View at Publisher · View at Google Scholar · View at Scopus M. L. Nelson and M. Y. Ismail, “The antibiotic and nonantibiotic tetracyclines,” in Comprehensive Medical Chemistry II , pp. 598–617, Elsevier, Amsterdam, The Netherlands, 2007. View at Google Scholar S. W. Anson, C. F. Chingnell, P. Roethling, and B. K. Cummings, “O 2 -photogenerated from aqueous solution of tetracycline antibiotics (pH 7.3) as evidenced by DMPO spin trapping and cytochrome C reduction,” Biochemical and Biophysical Research Communications , vol. 146, no. 3, pp. 1191–1195, 1987. View at Google Scholar A. S. Li, C. F. Chignell, and R. D. Hall, “Cutaneous phototoxicity of tetracycline antibiotics: generation of free radicals and singlet oxygen during photolysis as measured by spin-trapping and the phosphorescence of singlet molecular oxygen,” Photochemistry and Photobiology , vol. 46, no. 3, pp. 379–382, 1987. View at Publisher · View at Google Scholar · View at Scopus J. P. Martin, K. Colina, and N. Logsdon, “Role oxygen radicals in the phototoxicity of tetracyclines toward Escherichia coli B,” Journal of Bacteriology , vol. 169, no. 6, pp. 2516–2522, 1987. View at Google Scholar · View at Scopus T. Hasan, I. E. Kochevar, D. J. McAuliffe, B. S. Cooperman, and D. Abdulah, “Mechanism of tetracycline phototoxicity,” The Journal of Investigative Dermatology , vol. 83, no. 3, pp. 179–183, 1984. View at Publisher · View at Google Scholar · View at Scopus E. Ahler, W. J. Sullivan, A. Cass et al., “Doxycycline aAlters metabolism and proliferation of human cell line,” PLoS ONE , vol. 8, no. 5, Article ID e64561, 2013. View at Publisher · View at Google Scholar · View at Scopus A. Sigler, P. Schubert, W. Hillen, and M. Niederweis, “Permeation of tetracyclines through membranes of liposomes and Escherichia coli ,” European Journal of Biochemistry , vol. 267, no. 2, pp. 527–534, 2000. View at Publisher · View at Google Scholar · View at Scopus L. Brunton, B. Chabner, and B. Knollman, Goodman and Gilman's the Pharmacological Basis of Therapeutics , McGraw-Hill, New York, NY, USA, 10th edition, 2001. M. Nelson, W. Hillen, and R. A. Greenwald, Tetracyclines in Biology, Chemistry and Medicine , Birkhäuser, Basel, Switzerland, 2001. M. A. Khan, J. Mustafa, and J. Musarrat, “Mechanism of DNA strand breakage induced by photosensitized tetracycline-Cu(II) complex,” Mutation Research: Fundamental and Molecular Mechanisms of Mutagenesis , vol. 525, no. 1-2, pp. 109–119, 2003. View at Publisher · View at Google Scholar · View at Scopus D. Fuoco, “Classification framework and chemical biology of tetracycline-structure-based drugs,” Antibiotics , vol. 1, no. 1, pp. 1–13, 2012. View at Publisher · View at Google Scholar B. Carlotti, D. Fuoco, and F. Elisei, “Fast and ultrafast spectroscopic investigation of tetracycline derivatives in organic and aqueous media,” Physical Chemistry Chemical Physics , vol. 12, no. 48, pp. 15580–15591, 2010. View at Publisher · View at Google Scholar · View at Scopus N. J. Traynor, M. D. Barratt, W. W. Lovell, J. Ferguson, and N. K. Gibbs, “Comparison of an in vitro cellular phototoxicity model against controlled clinical trials of fluoroquinolone skin phototoxicity,” Toxicology in Vitro , vol. 14, no. 3, pp. 275–283, 2000. View at Publisher · View at Google Scholar · View at Scopus H. Spielmann, W. V. Lovell, and E. Holzle, “In vitro phototoxicity testing,” ATLA , vol. 22, pp. 314–348, 1994. View at Google Scholar J. M. Fagan, B. G. Sleczka, and I. Sohar, “Quantitation of oxidative damage to tissue proteins,” The International Journal of Biochemistry & Cell Biology , vol. 31, no. 7, pp. 751–757, 1999. View at Publisher · View at Google Scholar · View at Scopus M. R. Eftink and C. A. Ghiron, “Fluorescence quenching studies with proteins,” Analytical Biochemistry , vol. 114, no. 2, pp. 199–227, 1981. View at Publisher · View at Google Scholar · View at Scopus H. Terada, “Proposed partition mechanism of tetracycline,” Chemical and Pharmaceutical Bulletin , vol. 9, pp. 1965–1975, 1977. View at Google Scholar · View at Scopus A. K. Ghose and G. M. Crippen, “Atomic physicochemical parameters for three-dimensional-structure-directed quantitative structure-activity relationships. 2. Modeling dispersive and hydrophobic interactions,” Journal of Chemical Information and Computer Science , vol. 27, pp. 21–35, 1987. View at Google Scholar · View at Scopus F. Bahrami, D. L. Morris, and M. H. Pourgholami, “Tetracyclines: drugs with huge therapeutic potential,” Mini-Reviews in Medicinal Chemistry , vol. 12, no. 1, pp. 44–52, 2012. View at Publisher · View at Google Scholar · View at Scopus M. Bjellerup, T. Kjellstrom, and B. Ljunggren, “Influence of tetracycline phototoxicity on the growth of cultured human fibroblasts,” Journal of Investigative Dermatology , vol. 85, no. 6, pp. 573–574, 1985. View at Publisher · View at Google Scholar · View at Scopus A. M. Drucker and C. F. Rosen, “Drug-induced photosensitivity: culprit drugs, management and prevention,” Drug Safety , vol. 34, no. 10, pp. 821–837, 2011. View at Publisher · View at Google Scholar · View at Scopus E. L. Gelamo and M. Tabak, “Spectroscopic studies on the interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants,” Spectrochimica Acta—Part A: bolecular and Biomolecular Spectroscopy , vol. 56, no. 11, pp. 2255–2271, 2000. View at Publisher · View at Google Scholar · View at Scopus M. A. Khan, S. Muzammil, and J. Musarrat, “Differential binding of tetracyclines with serum albumin and induced structural alterations in drug-bound protein,” International Journal of Biological Macromolecules , vol. 30, no. 5, pp. 243–249, 2002. View at Publisher · View at Google Scholar · View at Scopus C. Jiang and L. Luo, “Spectrofluorimetric determination of human serum albumin using a doxycycline-europium probe,” Analytica Chimica Acta , vol. 506, no. 2, pp. 171–175, 2004. View at Publisher · View at Google Scholar · View at Scopus S. Bi, D. Song, Y. Tian, X. Zhou, Z. Liu, and H. Zhang, “Molecular spectroscopic study on the interaction of tetracyclines with serum albumins,” Spectrochimica Acta Part A , vol. 61, pp. 629–636, 2005. View at Publisher · View at Google Scholar M. I. Burgos, R. A. Fernández, M. S. Celej, L. I. Rossi, G. D. Fidelio, and S. A. Dassie, “Binding of the highly toxic tetracycline derivative, anhydrotetracycline, to bovine serum albumin,” Biological and Pharmaceutical Bulletin , vol. 34, no. 8, pp. 1301–1306, 2011. View at Publisher · View at Google Scholar · View at Scopus A. M. Layton and W. J. Cunliffe, “Phototoxic eruptions due to doxycycline—a dose-related phenomenon,” Clinical and Experimental Dermatology , vol. 18, no. 5, pp. 425–427, 1993. View at Publisher · View at Google Scholar · View at Scopus C. R. Shea, G. A. Olack, H. Morrison, N. Chen, and T. Hasan, “Phototoxicity of lumidoxycycline,” Journal of Investigative Dermatology , vol. 101, no. 3, pp. 329–333, 1993. View at Publisher · View at Google Scholar · View at Scopus A. Catalfo, G. Bracchitta, and G. de Guidi, “Role of aromatic amino acid tryptophan UVA-photoproducts in the determination of drug photosensitization mechanism: a comparison between methylene blue and naproxen,” Photochemical and Photobiological Sciences , vol. 8, no. 10, pp. 1467–1475, 2009. View at Publisher · View at Google Scholar · View at Scopus Q. H. Song, H. B. Wang, W. J. Tang, Q. X. Guo, and S. Q. Yu, “Model studies of the (6–4) photoproduct photoreactivation: efficient photosensitized splitting of thymine oxetane units by covalently linked tryptophan in high polarity solvents,” Organic and Biomolecular Chemistry , vol. 4, no. 2, pp. 291–298, 2006. View at Publisher · View at Google Scholar · View at Scopus J. A. M. Wiebe and D. E. Moore, “Oxidation photosensitized by tetracyclines,” Journal of Pharmaceutical Sciences , vol. 66, no. 2, pp. 186–189, 1977. View at Publisher · View at Google Scholar · View at Scopus H. Oka, Y. Ikai, N. Kawamura et al., “Photodecomposition products of tetracycline in aqueous solution,” Journal of Agricultural and Food Chemistry , vol. 37, no. 1, pp. 226–231, 1989. View at Publisher · View at Google Scholar · View at Scopus R. E. Drexel, G. Olack, C. Jones, G. N. Chmurny, R. Santini, and H. Morrison, “Lumitetracycline: a novel new tetracycline photoproduct,” Journal of Organic Chemistry , vol. 55, no. 8, pp. 2471–2478, 1990. View at Publisher · View at Google Scholar · View at Scopus G. Olack and H. Morrison, “Formation and characterization of lumitetracycline-type photoproducts from members of the tetracycline family,” Journal of Organic Chemistry , vol. 56, no. 16, pp. 4969–4971, 1991. View at Publisher · View at Google Scholar · View at Scopus M. M. Beliakova, S. I. Bessonov, B. M. Sergeyev, I. G. Smirnova, E. N. Dobrov, and A. M. Kopylov, “Rate of tetracycline photolysis during irradiation at 365 nm,” Biochemistry , vol. 68, no. 2, pp. 182–187, 2003. View at Publisher · View at Google Scholar · View at Scopus M. Addamo, V. Augugliaro, A. di Paola et al., “Removal of drugs in aqueous systems by photoassisted degradation,” Journal of Applied Electrochemistry , vol. 35, no. 7-8, pp. 765–774, 2005. View at Publisher · View at Google Scholar · View at Scopus J. J. López-Peñalver, M. Sánchez-Polo, C. V. Gómez-Pacheco, and J. Rivera-Utrilla, “Photodegradation of tetracyclines in aqueous solution by using UV and UV/H 2 O 2 oxidation processes,” Journal of Chemical Technology and Biotechnology , vol. 85, no. 10, pp. 1325–1333, 2010. View at Publisher · View at Google Scholar · View at Scopus A. Pena, L. P. Palilis, C. M. Lino, M. I. Silveira, and A. C. Calokerinos, “Determination of tetracycline and its major degradation products by chemiluminescence,” Analytica Chimica Acta , vol. 405, no. 1-2, pp. 51–56, 2000. View at Publisher · View at Google Scholar · View at Scopus Y. Chen, C. Hu, J. Qu, and M. Yang, “Photodegradation of tetracycline and formation of reactive oxygen species in aqueous tetracycline solution under simulated sunlight irradiation,” Journal of Photochemistry and Photobiology A: Chemistry , vol. 197, no. 1, pp. 81–87, 2008. View at Publisher · View at Google Scholar · View at Scopus A. Davies, G. O. Phillips, and A. G. Reid, “Photochemical oxidation of tetracycline in aqueous solution,” Journal of Chemical Spectroscopy Perkin II , pp. 369–375, 1979. View at Google Scholar · View at Scopus C. Chryssanthou, B. Graber, S. Mendelson, and G. Goldstein, “Increased blood-brain barrier permeability to tetracycline in rabbits under dysbaric conditions,” Undersea Biomedical Research , vol. 6, no. 4, pp. 319–328, 1979. View at Google Scholar · View at Scopus T. Rodgers and M. Rowland, “Physiologically based pharmacokinetic modelling 2: predicting the tissue distribution of acids, very weak bases, neutrals and zwitterions,” Journal of Pharmaceutical Sciences , vol. 95, no. 6, pp. 1238–1257, 2006. View at Publisher · View at Google Scholar · View at Scopus M. S. von Wittenau, “Some pharmacokinetic aspects of doxycycline metabolism in man.,” Chemotherapy , vol. 13, pp. 41–50, 1968. View at Publisher · View at Google Scholar · View at Scopus B. Ljunggren and M. Bjellerup, “Double blind cross-over studies on phototoxicity to three tetracycline derivatives in human volunteers,” Photodermatology , vol. 4, no. 6, pp. 281–287, 1987. View at Google Scholar · View at Scopus S. Sandberg, J. Glette, G. Hopen, and C. O. Solberg, “Doxycycline induced photodamage to Human Neutrophils and tryptophan,” Photochemistry and Photobiology , vol. 39, no. 1, pp. 43–48, 1984. View at Publisher · View at Google Scholar · View at Scopus R. Nilsson, G. Swanbeck, and G. Wennersten, “Primary mechanisms of erythrocyte photolysis induced by biological sensitizers and phototoxic drugs,” Photochemistry and Photobiology , vol. 22, no. 5, pp. 183–186, 1975. View at Publisher · View at Google Scholar · View at Scopus M. Bjellerup and B. Ljunggren, “Photohemolytic potency of tetracyclines,” Journal of Investigative Dermatology , vol. 84, no. 4, pp. 262–264, 1985. View at Publisher · View at Google Scholar · View at Scopus S. Jiao, S. Zheng, D. Yin, L. Wang, and L. Chen, “Aqueous photolysis of tetracycline and toxicity of photolytic products to luminescent bacteria,” Chemosphere , vol. 73, no. 3, pp. 377–382, 2008. 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