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Cite this: Photochem. Photobiol. Sci., 2014, 13, 621 Received 9th September 2013, Accepted 16th January 2014
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Anionic porphyrin as a new powerful cell death inducer of Tobacco Bright Yellow-2 cells† C. Riou,a C. A. Calliste,a A. Da Silva,b D. Guillaumot,a O. Rezazgui,a V. Sola and S. Leroy-Lhez*a
DOI: 10.1039/c3pp50315a www.rsc.org/pps
For the first time, the behaviour of tobacco cell suspensions submitted to four porphyrins was described. The potential killer effect of these photosensitizers on tobacco cells was evaluated. Biological results were correlated with photophysical properties and the reactive oxygen species production capacity of tested compounds. Surprisingly, the anionic free-base porphyrin showed the strongest phototoxic effect.
Introduction Biodiversity, environment preservation and health safety are the main challenges of agronomy. The search for new active herbicides that are plant-specific, non-toxic to wildlife, biodegradable by microorganisms and non-polluting towards groundwater is thus very important. Porphyrins and chlorins, naturally present in all living kingdoms, seem to be good candidates to meet these multiple expectations. Indeed, these molecular systems, being photoexcitable by sunlight, are able to induce cell death via the formation of reactive oxygen species (ROS) and present the advantage of having a very low toxicity in the absence of light.1 Until now, applications of such photosensitizers were essentially performed on carcinogen cells for photodynamic therapy (PDT)2 or on microorganisms as antimicrobial agents.3–8 Moreover, tested on flies or mosquito larvae, photoactivated porphyrins also seemed to have a potential insecticide effect.7–9 More recently, studies on plant organisms were realized to set up modifications of endogenous porphyrin biosynthesis by transgenesis.10–12 The authors showed that accumulation of protoporphyrin IX, the first photoexcited precursor of heme and chlorophylls, triggered lethal phenotypic alterations (growth inhibition, browning leaves, and
desiccation).11,12 Altogether, these results suggest that hydrosoluble porphyrins could pass over different kinds of cell walls even if they are composed of various molecules such as peptidoglycans for bacteria, mannan, glucan or chitin for insects and fungi. Like bacteria and fungi, plant cells are surrounded by a cell wall. Nevertheless, the chemical structure of the plant primary cell wall is largely different from that of a microorganism as it is mainly composed of polysaccharides such as cellulose, hemicelluloses and pectins.13 Indeed, the plant cell wall is a dynamic structure playing multiple roles in plant growth and development such as physical barrier, elongation, orientation of cell division, water exchange, and cell to cell communication.14 Taking into account phototoxic effects of porphyrins on microorganisms which are protected by a cell wall, we supposed that porphyrins could also be photoactivated in plant cells and trigger cell death. Besides, a study performed on Allium cepa roots showed that cationic porphyrins were able to enter cells and induce DNA photodamage.15 Thus, our aim was to use exogenous hydrosoluble porphyrins as biodegradable herbicides. For that purpose, we studied, both in solution and on Tobacco Bright Yellow-2 (TBY-2) cell suspensions, four hydrosoluble porphyrins CP, CP-Zn, AP, and AP-Zn (Scheme 1). Indeed these molecular systems were chosen to get further insight into the photochemical mechanism and response induced in TBY-2 cells after their photoactivation. The nature of the produced ROS and their production efficiency as their absorption and emission properties will be correlated with their toxicity under light exposure. Photo-bleaching and/or photo-transformation of the 4 compounds will also be discussed.
Results and discussion Synthesis of AP-Zn
a
Université de Limoges, Laboratoire de Chimie des Substances Naturelles, EA 1069, 123 avenue Albert Thomas, 87060 Limoges, France. E-mail: [email protected] b Université de Limoges, Laboratoire de Génétique Moléculaire Animale, UMR-INRA 1061, 123 avenue Albert Thomas, 87060 Limoges, France † Electronic supplementary information (ESI) available: Materials and methods and spectroscopic data for all porphyrins studied. See DOI: 10.1039/c3pp50315a
This journal is © The Royal Society of Chemistry and Owner Societies 2014
As metallated anionic porphyrin AP-Zn was the only non-commercial product used in this study, we synthesized it in order to be able to study both the photosensitizer charge and the metallation effect on TBY-2 cells. AP-Zn was therefore prepared according to the classical porphyrin metallation procedure.16
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Scheme 1
Photochemical & Photobiological Sciences
Structure of free-base or Zn-metallated cationic (CP and CP-Zn) and anionic (AP and AP-Zn) porphyrins.
Treatment of AP with zinc(II) acetate in water resulted, after dialysis, in the obtainment of AP-Zn in quantitative yield. The reaction was monitored by UV-visible spectroscopy as freebase, and metallated porphyrins exhibit different typical spectral features in the visible range (Q bands). Spectroscopic properties and photostability The fluorescence emission and UV-visible spectral data of the four studied porphyrins in water are listed in Table 1. Their fluorescence emission spectra were found to be independent of the excitation wavelength. Fluorescence excitation spectra matched the absorption profile over the entire wavelength range. As expected, all four compounds presented a porphyrin typical weak emission (all quantum yields being inferior to 0.05 at room temperature) centred from 606 nm to 680 nm, depending on the considered porphyrin. Absorption spectra of CP and AP in water showed the typical Soret and Q bands characteristic of free-base porphyrin derivatives. The relative
Table 1 Selected photophysical data in water for CP, CP-Zn, AP, and AP-Zn and their photostability in TBY-2 growth medium after irradiation with white light (95 µE s−1 m−2) for different periods of time (20 °C; conc. εIII ≈ εII > εI) whereas an etio-type spectrum (εIV > εIII > εII > εI) was recorded for AP. Moreover, neither aggregates nor protonation17 of AP were observed at the concentration used for biological experiments (3.5 µmol L−1) in water or TBY-2 growth medium. As expected, metallation by zinc led to a modification of UV-visible spectra of CP-Zn and AP-Zn. Indeed, a slight bathochromic shift of the Soret band and a decrease in the number of Q bands were observed. An absorption coefficient increase, in particular regarding the Soret band, was also observed. In order to check the photostability of the different porphyrins, their absorption spectra were monitored for different irradiation times under the same conditions as TBY-2 cells (light, growth medium and concentration). Our attention was focused on both the variation of the intensity of the Soret band and the potential change in the UV-visible spectrum profile. The results of the photostability assays are summarized in Table 1. In general, with the exception of AP-Zn, which also showed the lowest photostability (22.4% of the initial Soret band absorption after 1 hour of irradiation and only 1.8% after 4 hours), there were no changes in the absorbance spectra, indicating that the other three porphyrins did not undergo phototransformation but only photobleaching. We also noticed that metallated porphyrins were less photostable than the free-base ones and that cationic porphyrins were the most stable.
Compd
λabs
ε
λem max
ϕfluo
0
1
4
ROS production
CP
423 519 555 586 640 437 565 608 414 516 553 582 636 422 557 596
153 200 9500 4200 4100 1000 268 100 21 700 6800 197 800 6500 3350 3750 3700 434 700 13 700 5500
680
0.016
100
55.7
51
634
0.025
100
74.3
66.5
644
0.046
100
39.6
31.2
606
0.03
100
22.4
1.8
Photoactivation of photosensitizers might undergo a photoinduced electron transfer (Type I process), generating radicals such as the superoxide anion (O2•−) and the hydroxyl radical (•OH), or energy transfer to dioxygen (Type II process), producing singlet oxygen (1O2).18 Thus, we investigated the kind of mechanism involved in phototoxicity. For that purpose, we used Electron Paramagnetic Resonance (EPR) spectroscopy to detect ROS such as O2•− and 1O2. As these species are shortlived ones, experiments were performed in the presence of specific scavengers. We estimated the production of singlet oxygen for CP, CP-Zn and AP, photostability of AP-Zn being very low, thanks to 2,2,6,6-tetramethyl-4-piperidone (TEMP), which, in the presence of singlet oxygen, was transformed into TEMPO, the corresponding nitroxide radical, detectable by EPR.19,20 The results obtained for the standard reaction
CP-Zn AP
AP-Zn
λabs: main absorption (nm). ε: molar absorption coefficient (L mol−1 cm−1). λem max: fluorescence emission maxima (nm). ϕfluo: fluorescence quantum yield.
622 | Photochem. Photobiol. Sci., 2014, 13, 621–625
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Fig. 1 (A) EPR signal of TEMPO generation by irradiation of CP, CP-Zn and AP. (B) EPR signal of DMPO-OOH generated by irradiation of CP or AP ( porphyrins conc. = 40 µmol L−1 for experiments A; values represent the mean ± S.D. obtained from 3 independent experiments for A; experiments were performed as described in ESI†).
mixture (12.5 mM TEMP and 40 µM porphyrin solution in 0.01 M phosphate buffer, pH 7.4) exposed to white light (270 µE s−1 m−2) for different periods of time are shown in Fig. 1A. No variation of the initial EPR signal was observed when the reaction mixture was measured without light irradiation or in the absence of a photosensitizer. As can be outlined, both freebase porphyrins CP and AP exhibited the best singlet oxygen production. Indeed, under the experimental conditions, even if initial singlet oxygen generation was very good, it seemed that CP-Zn was very rapidly degraded, probably by 1O2 itself. As high singlet oxygen yield production is one of the conditions for efficient phototoxicity, CP and AP could be considered as potential good candidates for application as photoherbicides. For these two compounds, production of the superoxide radical was also checked. The reaction between molecular oxygen and CP or AP was thus studied in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) which leads to a DMPO–OOH adduct with the characteristic EPR spectrum.21 As the signal for the DMPO–OOH was not observed in aqueous solution,18 experiments were conducted in DMSO. Both CP and AP produced superoxide anions, evidencing that
a type II pathway could contribute to the photosensitization process (Fig. 1B). It is worth noting that CP appeared to be really more efficient at superoxide anion generation (more than 240 times) than AP. Biological experiments All porphyrins were tested on TBY-2 cell suspensions and the percentage of cell viability was determined after each treatment (Fig. 2A). After 3 h incubation with porphyrins under dark conditions, exponential phase cells were illuminated for 5 h and put back into the dark for 18 hours. Aliquots of cells were then stained with Trypan blue and counted under a photonic microscope. Results obtained with control cells (without porphyrin and with porphyrin under the dark) showed that porphyrins without light exposure did not induce cell death of TBY-2 cell suspensions and thus were not cytotoxic for plant cells. When tested at 3.5 μM, after illumination, all porphyrins of interest significantly induced TBY-2 cell death although a weak photon intensity was delivered to cells (95 μE s−1 m−2) compared to what it was performed on bacteria and fungi.22–25 Therefore even at low light intensity, the four compounds were
Fig. 2 Effects of the four different porphyrins after 5 h photoactivation on TBY-2 cells: (A) percentage of cell survival and (B) H2O2 production in plant cells (H2O referred to control cells without porphyrin; cells were treated as described in ESI;† porphyrins conc. = 3.5 µmol L−1; cells were kept under dark conditions (dark box) or under light for 5 hours (grey box); values represent the mean ± SEM obtained from independent experiments (10 to 19 depending on the group considered for A and 4 for B; ***P < 0.001; *P < 0.05; NS: not significant, i.e. P > 0.05).
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able to significantly induce TBY-2 cell death. We hypothesized that porphyrins could be localized very close to and/or inside cells and their photoactivation generated ROS, as was evidenced by EPR experiments and DAB staining (Fig. 2B). Surprisingly, the most efficient photosensitizer was AP, inducing more than 90% of cell death whereas the other porphyrins induced cell death to a lesser extent (around 40% of cell death). Indeed, to the best of our knowledge and by extrapolation from mammal and insect cells to microorganisms,1,3–8,15,22–25 cationic porphyrins were always the most efficient on living systems. Moreover, AP is neither the best producer of ROS (singlet oxygen and superoxide anions in particular), as shown by EPR measurement, nor the most photostable, in contrast to CP (Table 1 and Fig. 1). Thus, this result remained difficult to explain. In regard to the composition of the plant cell wall which is rich in pectins and could be negatively charged,13 we supposed that CP could be trapped into the cell wall. Its photoactivation and subsequent production of ROS would thus be prevented. This would not be the case for AP which could cross the cell wall and be localized either in the apoplasm and/ or the cytoplasm. Further investigations are necessary to test this hypothesis, such as direct localization of AP into TBY-2 cells by confocal microscopy. Results obtained for AP-Zn might be due to its very low photostability (Table 1). Indeed, even if AP-Zn had the same kind of interaction with TBY-2 cells as AP, its instability made its concentration in cells not sufficient to induce a similar percentage of cell death. Whereas AP-Zn was less efficient in inducing cell death compared to its homolog AP, CP and CP-Zn gave the same percentage of cell death after photoactivation (Fig. 2A). This result suggested that the photoactivation efficiency of both cationic porphyrins was not linked to the metal presence. Furthermore, we stained aliquots of plant cells with DAB which is used to detect H2O2 in cells. Indeed, photoactivated porphyrins led to O2•− production which was partially transformed by TBY-2 cells into H2O2, a less toxic species for cells.26 AP treated cells showed an almost twice higher percentage of DAB stained cells (ca. 90%) than cells treated with the other three porphyrins (ca. 50%) (Fig. 2B). This suggested a high production of H2O2 in AP treated cells well correlated to DNA fragmentation and a high percentage of cell death (Fig. 2A and 3). Thus H2O2 and other ROS productions were a consequence of porphyrin photoactivation as shown in other living cells. In plants, the application of exogenous aminolevulinic acid (the first porphyrin precursor) showed plant bleaching and accumulation of protoporphyrin IX, the first photosensitive molecule of the plant tetrapyrrole pathway.27 In the same way, herbicides targeting plant endogenous protoporphyrinogen oxidase also led to leaf burning, desiccation and growth inhibition.11,28 The same kind of results was observed with transgenic rice overexpressing protoporphyrinogen IX oxidase.10,12 It was proposed that exposure to light caused formation of singlet oxygen and other oxidative species, resulting in membrane disruption and subsequent cell death.26,29 Altogether, these results allowed us to establish a link between porphyrin photoactivation, ROS production and TBY-2 cell death. Moreover, DNA
624 | Photochem. Photobiol. Sci., 2014, 13, 621–625
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Fig. 3 DNA fragmentation in TBY-2 cells treated with porphyrins after photoactivation (DNA isolation was performed as described in ESI;† porphyrins conc. = 3.5 µmol L−1; cells were kept under dark conditions (D) or under light for 5 hours (L); the DNA marker was loaded on lanes 5 and 8).
fragmentation observed only for AP treated cells strongly suggested that cell death was linked to apoptosis rather than necrosis. However AP being less effective than CP in terms of singlet oxygen and superoxide anion production, accumulation of H2O2 in AP treated cells might be due to inhibition of enzymes involved in the detoxification pathway such as peroxidases and catalases that should be measured.
Conclusions In this preliminary study, we showed that TBY-2 cells were extremely sensitive to photoexcitation of tested porphyrins, especially the anionic one, AP, leading to high production of hydrogen peroxide and apoptosis. This result was partially surprising in regard to the highest photocytotoxicity of cationic porphyrins when tested on other living systems, and made AP a potential candidate as a photoherbicide. Further investigations are needed to study the mechanisms involved in plant cell death. To gain insight into the interaction between plant cells and porphyrins, the specific localization of photosensitizers in cells will be very important. As AP was a weak fluorescent molecule, it remained difficult to directly localize it into cells. Thus, synthesis of new fluorescent tagged anionic porphyrins is currently in progress in our laboratory to resolve this problem.
Acknowledgements We thank the French National Research Agency (ANR PorphyPlant) for financial support.
Notes and references 1 V. Sarrazy, G. Garcia, J.-P. MBakidi, C. Le Morvan, G. Bégaud-Grimaud, R. Granet, V. Sol and P. Krausz,
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3 4 5
6
7
8
9
10
11
12
13 14
15
Photodynamic effects of porphyrins polyamine conjugates in human breast cancer and keratinocyte cell lines, J. Photochem. Photobiol., B, 2011, 103, 201–206. S. Yano, S. Hirohara, M. Obata, Y. Hagiya, S.-I. Ogura, A. Ikeda, H. Kataoka, M. Tanaka and T. Joh, Current states and future views in photodynamic therapy, J. Photochem. Photobiol., C, 2011, 12, 46–67. G. Jori and B. Brown, Photosensitized inactivation of microorganisms, Photochem. Photobiol. Sci., 2004, 3, 403–405. T. Maisch, Anti-microbial photodynamic therapy: useful in the future?, Lasers Med. Sci., 2007, 22, 83–91. C. Ringot, N. Saad, R. Granet, P. Bressollier, V. Sol and P. Krausz, Meso-functionalized aminoporphyrins as efficient agents for photo-antibacterial surfaces, J. Porphyrins Phthalocyanines, 2010, 14, 925–931. C. Ringot, V. Sol, M. Barrière, N. Saad, P. Bressollier, R. Granet, P. Couleaud, C. Frochot and P. Krausz, Triazinyl porphyrin-based photoactive cotton fabrics: preparation, characterization and antibacterial activity, Biomacromolecules, 2011, 12, 1716–1723. T. Ben Amor and G. Jori, Sunlight-activated insecticides: Historical background and mechanisms of phototoxic activity, Insect Biochem. Mol. Biol., 2000, 30, 915–925. B. Dondji, S. Duchon, A. Diabate, J.-P. Herve, V. Corbel, J.-M. Hougard, R. Santus and J. Schrevel, Assessment of Laboratory and Field Assays of Sunlight-Induced Killing of Mosquito Larvae by Photosensitizers, J. Med. Entomol., 2005, 42, 652–656. L. Lucantoni, M. Magaraggia, G. Lupidi, R. Ouedraogo, O. Coppellotti, F. Esposito, C. Fabris, G. Jori and A. Habluetzel, Novel, Meso-substituted cationic porphyrin molecule for photo mediated larval control of the dengue vector Aedes aegypti, PLoS Negl. Trop. Dis., 2011, 5, 1434– 1445. Y. Kuk, H. Lee, J. Chung, K. Kim, S. Lee, S. Ha, K. Back and J. Guh, Expression of a Bacillus subtilis protoporphyrinogen oxidase gene in rice plants reduces sensitivity to peroxidizing herbicides, Biol. Plant., 2005, 49, 577–583. X. Li and D. Nicholl, Development of PPO inhibitor-resistant cultures and crops, Pest Manage. Sci., 2005, 61, 277–285. S. Jung, H. Lee, Y. Lee, K. Kang, Y. S. Kim, B. Grimm and K. Back, Toxic tetrapyrrole accumulation in protoporphyrinogen IX oxidase-overexpressing transgenic rice plants, Plant Mol. Biol., 2008, 67, 535–546. M. C. Jarvis, Plant cell walls: supramolecular assemblies, Food Hydrocolloids, 2011, 25, 257–262. C. Somerville, S. Bauer, G. Brininstool, M. Facette, T. Hamann, J. Milne, E. Osborne, A. Paredez, S. Persson, T. Raab, S. Vorwerk and H. Youngs, Toward a system approach to understanding plant cell walls, Science, 2004, 306, 2206–2211. A. Villanueva, M. Cañete and M. J. Hazen, Uptake and DNA photodamage induced in plant cells in vivo by two cationic porphyrins, Mutagenesis, 1989, 4, 157–159.
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16 A. Bardat, V. Gloaguen, V. Sol and P. Krausz, Aqueous extraction of glucuronoxylans from chestnut wood: new strategy for lignin oxidation using phthalocyanine or porphyrin/H2O2 system, Bioresour. Technol., 2012, 101, 6538–6543. 17 J. V. Hollingsworth, A. J. Richard, M. Graça, H. Vicente and P. S. Russo, Characterization of the Self-Assembly of mesoTetra(4-sulfonatophenyl)porphyrin (H2TPPS4–) in Aqueous Solutions, Biomacromolecules, 2012, 13, 60–72. 18 K. Ergaieg, M. Chevanne, J. Cillard and R. Seux, Involvement of both Type I and Type II mechanisms in Grampositive and Gram-negative bacteria photosensitization by a meso-substituted cationic porphyrins, Sol. Energy, 2008, 82, 1107–1117. 19 Y. Lion, M. Delmelle and A. van de Vorst, New method of detecting singlet oxygen production, Nature, 1976, 263, 442–443. 20 J. Moan and E. Wold, Detection of singlet oxygen production by ESR, Nature, 1979, 279, 450–451. 21 G. R. Buettner and L. W. Oberley, Considerations in the spin trapping of superoxide and hydroxyl radicals in aqueous systems using 5,5-dimethyl-1-pyrroline-1-oxide, Biochem. Biophys. Res. Commun., 1978, 83, 69–74. 22 M. Drabkova, B. Marsalek and W. Admiral, Photodynamic therapy against cyanobacteria, Environ. Toxicol., 2007, 22, 112–115. 23 M. Merchat, G. Bertoloni, P. Giacomoni, A. Villanueva and G. Jori, Meso-substituted cationic porphyrins as efficient photosensitizers of gram-positive and gram-negative bacteria, J. Photochem. Photobiol., 1996, 32, 153–157. 24 M. P. Cormick, M. G. Alavrez, M. Rovera and E. N. Durantini, Photodynamic inactivation of Candida albicans sensitized by tri- and tetra-cationic porphyrin derivatives, Eur. J. Med. Chem., 2009, 44, 1592–1599. 25 V. Carré, O. Gaud, I. Sylvain, O. Bourdon, M. Spiro, J. Blais, R. Granet, P. Krausz and M. Guilloton, Fungicidal properties of meso-arylglycosylporphyrins: influence of sugar substituents on photoinduced damage in the yeast Saccharomyces cerevisiae, J. Photochem. Photobiol., 1999, 48, 57–62. 26 M. C. De Pinto, A. Paradiso, P. Leonetti and L. De Gara, Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defense and cell death, Plant J., 2006, 48, 784–795. 27 H. Matsumoto, Y. Tanida and K. Ishizuka, Porphyrin Intermediates in Herbicidal Action of d-Aminolevulinic Acid on Duckweed (Lemna paucicostata Hegelm.), Pestic. Biochem. Physiol., 1994, 48, 214–221. 28 J. M. Jacobs and N. Jacobs, Porphyrin accumulation and export by isolated barley (Hordeum vulgare) plastids (Effect of Diphenyl Ether Herbicides), J. Plant Physiol., 1993, 101, 1181–1187. 29 M. Matringe and R. Scalla, Studies on the mode of action of acifluorfen-methyl in nonchlorophyllus soybean cells, Plant Physiol., 1998, 86, 619–622.
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Cite this: Photochem. Photobiol. Sci., 2014, 13, 621 Received 9th September 2013, Accepted 16th January 2014
View Journal | View Issue
Anionic porphyrin as a new powerful cell death inducer of Tobacco Bright Yellow-2 cells† C. Riou,a C. A. Calliste,a A. Da Silva,b D. Guillaumot,a O. Rezazgui,a V. Sola and S. Leroy-Lhez*a
DOI: 10.1039/c3pp50315a www.rsc.org/pps
For the first time, the behaviour of tobacco cell suspensions submitted to four porphyrins was described. The potential killer effect of these photosensitizers on tobacco cells was evaluated. Biological results were correlated with photophysical properties and the reactive oxygen species production capacity of tested compounds. Surprisingly, the anionic free-base porphyrin showed the strongest phototoxic effect.
Introduction Biodiversity, environment preservation and health safety are the main challenges of agronomy. The search for new active herbicides that are plant-specific, non-toxic to wildlife, biodegradable by microorganisms and non-polluting towards groundwater is thus very important. Porphyrins and chlorins, naturally present in all living kingdoms, seem to be good candidates to meet these multiple expectations. Indeed, these molecular systems, being photoexcitable by sunlight, are able to induce cell death via the formation of reactive oxygen species (ROS) and present the advantage of having a very low toxicity in the absence of light.1 Until now, applications of such photosensitizers were essentially performed on carcinogen cells for photodynamic therapy (PDT)2 or on microorganisms as antimicrobial agents.3–8 Moreover, tested on flies or mosquito larvae, photoactivated porphyrins also seemed to have a potential insecticide effect.7–9 More recently, studies on plant organisms were realized to set up modifications of endogenous porphyrin biosynthesis by transgenesis.10–12 The authors showed that accumulation of protoporphyrin IX, the first photoexcited precursor of heme and chlorophylls, triggered lethal phenotypic alterations (growth inhibition, browning leaves, and
desiccation).11,12 Altogether, these results suggest that hydrosoluble porphyrins could pass over different kinds of cell walls even if they are composed of various molecules such as peptidoglycans for bacteria, mannan, glucan or chitin for insects and fungi. Like bacteria and fungi, plant cells are surrounded by a cell wall. Nevertheless, the chemical structure of the plant primary cell wall is largely different from that of a microorganism as it is mainly composed of polysaccharides such as cellulose, hemicelluloses and pectins.13 Indeed, the plant cell wall is a dynamic structure playing multiple roles in plant growth and development such as physical barrier, elongation, orientation of cell division, water exchange, and cell to cell communication.14 Taking into account phototoxic effects of porphyrins on microorganisms which are protected by a cell wall, we supposed that porphyrins could also be photoactivated in plant cells and trigger cell death. Besides, a study performed on Allium cepa roots showed that cationic porphyrins were able to enter cells and induce DNA photodamage.15 Thus, our aim was to use exogenous hydrosoluble porphyrins as biodegradable herbicides. For that purpose, we studied, both in solution and on Tobacco Bright Yellow-2 (TBY-2) cell suspensions, four hydrosoluble porphyrins CP, CP-Zn, AP, and AP-Zn (Scheme 1). Indeed these molecular systems were chosen to get further insight into the photochemical mechanism and response induced in TBY-2 cells after their photoactivation. The nature of the produced ROS and their production efficiency as their absorption and emission properties will be correlated with their toxicity under light exposure. Photo-bleaching and/or photo-transformation of the 4 compounds will also be discussed.
Results and discussion Synthesis of AP-Zn
a
Université de Limoges, Laboratoire de Chimie des Substances Naturelles, EA 1069, 123 avenue Albert Thomas, 87060 Limoges, France. E-mail: [email protected] b Université de Limoges, Laboratoire de Génétique Moléculaire Animale, UMR-INRA 1061, 123 avenue Albert Thomas, 87060 Limoges, France † Electronic supplementary information (ESI) available: Materials and methods and spectroscopic data for all porphyrins studied. See DOI: 10.1039/c3pp50315a
This journal is © The Royal Society of Chemistry and Owner Societies 2014
As metallated anionic porphyrin AP-Zn was the only non-commercial product used in this study, we synthesized it in order to be able to study both the photosensitizer charge and the metallation effect on TBY-2 cells. AP-Zn was therefore prepared according to the classical porphyrin metallation procedure.16
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Scheme 1
Photochemical & Photobiological Sciences
Structure of free-base or Zn-metallated cationic (CP and CP-Zn) and anionic (AP and AP-Zn) porphyrins.
Treatment of AP with zinc(II) acetate in water resulted, after dialysis, in the obtainment of AP-Zn in quantitative yield. The reaction was monitored by UV-visible spectroscopy as freebase, and metallated porphyrins exhibit different typical spectral features in the visible range (Q bands). Spectroscopic properties and photostability The fluorescence emission and UV-visible spectral data of the four studied porphyrins in water are listed in Table 1. Their fluorescence emission spectra were found to be independent of the excitation wavelength. Fluorescence excitation spectra matched the absorption profile over the entire wavelength range. As expected, all four compounds presented a porphyrin typical weak emission (all quantum yields being inferior to 0.05 at room temperature) centred from 606 nm to 680 nm, depending on the considered porphyrin. Absorption spectra of CP and AP in water showed the typical Soret and Q bands characteristic of free-base porphyrin derivatives. The relative
Table 1 Selected photophysical data in water for CP, CP-Zn, AP, and AP-Zn and their photostability in TBY-2 growth medium after irradiation with white light (95 µE s−1 m−2) for different periods of time (20 °C; conc. εIII ≈ εII > εI) whereas an etio-type spectrum (εIV > εIII > εII > εI) was recorded for AP. Moreover, neither aggregates nor protonation17 of AP were observed at the concentration used for biological experiments (3.5 µmol L−1) in water or TBY-2 growth medium. As expected, metallation by zinc led to a modification of UV-visible spectra of CP-Zn and AP-Zn. Indeed, a slight bathochromic shift of the Soret band and a decrease in the number of Q bands were observed. An absorption coefficient increase, in particular regarding the Soret band, was also observed. In order to check the photostability of the different porphyrins, their absorption spectra were monitored for different irradiation times under the same conditions as TBY-2 cells (light, growth medium and concentration). Our attention was focused on both the variation of the intensity of the Soret band and the potential change in the UV-visible spectrum profile. The results of the photostability assays are summarized in Table 1. In general, with the exception of AP-Zn, which also showed the lowest photostability (22.4% of the initial Soret band absorption after 1 hour of irradiation and only 1.8% after 4 hours), there were no changes in the absorbance spectra, indicating that the other three porphyrins did not undergo phototransformation but only photobleaching. We also noticed that metallated porphyrins were less photostable than the free-base ones and that cationic porphyrins were the most stable.
Compd
λabs
ε
λem max
ϕfluo
0
1
4
ROS production
CP
423 519 555 586 640 437 565 608 414 516 553 582 636 422 557 596
153 200 9500 4200 4100 1000 268 100 21 700 6800 197 800 6500 3350 3750 3700 434 700 13 700 5500
680
0.016
100
55.7
51
634
0.025
100
74.3
66.5
644
0.046
100
39.6
31.2
606
0.03
100
22.4
1.8
Photoactivation of photosensitizers might undergo a photoinduced electron transfer (Type I process), generating radicals such as the superoxide anion (O2•−) and the hydroxyl radical (•OH), or energy transfer to dioxygen (Type II process), producing singlet oxygen (1O2).18 Thus, we investigated the kind of mechanism involved in phototoxicity. For that purpose, we used Electron Paramagnetic Resonance (EPR) spectroscopy to detect ROS such as O2•− and 1O2. As these species are shortlived ones, experiments were performed in the presence of specific scavengers. We estimated the production of singlet oxygen for CP, CP-Zn and AP, photostability of AP-Zn being very low, thanks to 2,2,6,6-tetramethyl-4-piperidone (TEMP), which, in the presence of singlet oxygen, was transformed into TEMPO, the corresponding nitroxide radical, detectable by EPR.19,20 The results obtained for the standard reaction
CP-Zn AP
AP-Zn
λabs: main absorption (nm). ε: molar absorption coefficient (L mol−1 cm−1). λem max: fluorescence emission maxima (nm). ϕfluo: fluorescence quantum yield.
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Fig. 1 (A) EPR signal of TEMPO generation by irradiation of CP, CP-Zn and AP. (B) EPR signal of DMPO-OOH generated by irradiation of CP or AP ( porphyrins conc. = 40 µmol L−1 for experiments A; values represent the mean ± S.D. obtained from 3 independent experiments for A; experiments were performed as described in ESI†).
mixture (12.5 mM TEMP and 40 µM porphyrin solution in 0.01 M phosphate buffer, pH 7.4) exposed to white light (270 µE s−1 m−2) for different periods of time are shown in Fig. 1A. No variation of the initial EPR signal was observed when the reaction mixture was measured without light irradiation or in the absence of a photosensitizer. As can be outlined, both freebase porphyrins CP and AP exhibited the best singlet oxygen production. Indeed, under the experimental conditions, even if initial singlet oxygen generation was very good, it seemed that CP-Zn was very rapidly degraded, probably by 1O2 itself. As high singlet oxygen yield production is one of the conditions for efficient phototoxicity, CP and AP could be considered as potential good candidates for application as photoherbicides. For these two compounds, production of the superoxide radical was also checked. The reaction between molecular oxygen and CP or AP was thus studied in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) which leads to a DMPO–OOH adduct with the characteristic EPR spectrum.21 As the signal for the DMPO–OOH was not observed in aqueous solution,18 experiments were conducted in DMSO. Both CP and AP produced superoxide anions, evidencing that
a type II pathway could contribute to the photosensitization process (Fig. 1B). It is worth noting that CP appeared to be really more efficient at superoxide anion generation (more than 240 times) than AP. Biological experiments All porphyrins were tested on TBY-2 cell suspensions and the percentage of cell viability was determined after each treatment (Fig. 2A). After 3 h incubation with porphyrins under dark conditions, exponential phase cells were illuminated for 5 h and put back into the dark for 18 hours. Aliquots of cells were then stained with Trypan blue and counted under a photonic microscope. Results obtained with control cells (without porphyrin and with porphyrin under the dark) showed that porphyrins without light exposure did not induce cell death of TBY-2 cell suspensions and thus were not cytotoxic for plant cells. When tested at 3.5 μM, after illumination, all porphyrins of interest significantly induced TBY-2 cell death although a weak photon intensity was delivered to cells (95 μE s−1 m−2) compared to what it was performed on bacteria and fungi.22–25 Therefore even at low light intensity, the four compounds were
Fig. 2 Effects of the four different porphyrins after 5 h photoactivation on TBY-2 cells: (A) percentage of cell survival and (B) H2O2 production in plant cells (H2O referred to control cells without porphyrin; cells were treated as described in ESI;† porphyrins conc. = 3.5 µmol L−1; cells were kept under dark conditions (dark box) or under light for 5 hours (grey box); values represent the mean ± SEM obtained from independent experiments (10 to 19 depending on the group considered for A and 4 for B; ***P < 0.001; *P < 0.05; NS: not significant, i.e. P > 0.05).
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able to significantly induce TBY-2 cell death. We hypothesized that porphyrins could be localized very close to and/or inside cells and their photoactivation generated ROS, as was evidenced by EPR experiments and DAB staining (Fig. 2B). Surprisingly, the most efficient photosensitizer was AP, inducing more than 90% of cell death whereas the other porphyrins induced cell death to a lesser extent (around 40% of cell death). Indeed, to the best of our knowledge and by extrapolation from mammal and insect cells to microorganisms,1,3–8,15,22–25 cationic porphyrins were always the most efficient on living systems. Moreover, AP is neither the best producer of ROS (singlet oxygen and superoxide anions in particular), as shown by EPR measurement, nor the most photostable, in contrast to CP (Table 1 and Fig. 1). Thus, this result remained difficult to explain. In regard to the composition of the plant cell wall which is rich in pectins and could be negatively charged,13 we supposed that CP could be trapped into the cell wall. Its photoactivation and subsequent production of ROS would thus be prevented. This would not be the case for AP which could cross the cell wall and be localized either in the apoplasm and/ or the cytoplasm. Further investigations are necessary to test this hypothesis, such as direct localization of AP into TBY-2 cells by confocal microscopy. Results obtained for AP-Zn might be due to its very low photostability (Table 1). Indeed, even if AP-Zn had the same kind of interaction with TBY-2 cells as AP, its instability made its concentration in cells not sufficient to induce a similar percentage of cell death. Whereas AP-Zn was less efficient in inducing cell death compared to its homolog AP, CP and CP-Zn gave the same percentage of cell death after photoactivation (Fig. 2A). This result suggested that the photoactivation efficiency of both cationic porphyrins was not linked to the metal presence. Furthermore, we stained aliquots of plant cells with DAB which is used to detect H2O2 in cells. Indeed, photoactivated porphyrins led to O2•− production which was partially transformed by TBY-2 cells into H2O2, a less toxic species for cells.26 AP treated cells showed an almost twice higher percentage of DAB stained cells (ca. 90%) than cells treated with the other three porphyrins (ca. 50%) (Fig. 2B). This suggested a high production of H2O2 in AP treated cells well correlated to DNA fragmentation and a high percentage of cell death (Fig. 2A and 3). Thus H2O2 and other ROS productions were a consequence of porphyrin photoactivation as shown in other living cells. In plants, the application of exogenous aminolevulinic acid (the first porphyrin precursor) showed plant bleaching and accumulation of protoporphyrin IX, the first photosensitive molecule of the plant tetrapyrrole pathway.27 In the same way, herbicides targeting plant endogenous protoporphyrinogen oxidase also led to leaf burning, desiccation and growth inhibition.11,28 The same kind of results was observed with transgenic rice overexpressing protoporphyrinogen IX oxidase.10,12 It was proposed that exposure to light caused formation of singlet oxygen and other oxidative species, resulting in membrane disruption and subsequent cell death.26,29 Altogether, these results allowed us to establish a link between porphyrin photoactivation, ROS production and TBY-2 cell death. Moreover, DNA
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Fig. 3 DNA fragmentation in TBY-2 cells treated with porphyrins after photoactivation (DNA isolation was performed as described in ESI;† porphyrins conc. = 3.5 µmol L−1; cells were kept under dark conditions (D) or under light for 5 hours (L); the DNA marker was loaded on lanes 5 and 8).
fragmentation observed only for AP treated cells strongly suggested that cell death was linked to apoptosis rather than necrosis. However AP being less effective than CP in terms of singlet oxygen and superoxide anion production, accumulation of H2O2 in AP treated cells might be due to inhibition of enzymes involved in the detoxification pathway such as peroxidases and catalases that should be measured.
Conclusions In this preliminary study, we showed that TBY-2 cells were extremely sensitive to photoexcitation of tested porphyrins, especially the anionic one, AP, leading to high production of hydrogen peroxide and apoptosis. This result was partially surprising in regard to the highest photocytotoxicity of cationic porphyrins when tested on other living systems, and made AP a potential candidate as a photoherbicide. Further investigations are needed to study the mechanisms involved in plant cell death. To gain insight into the interaction between plant cells and porphyrins, the specific localization of photosensitizers in cells will be very important. As AP was a weak fluorescent molecule, it remained difficult to directly localize it into cells. Thus, synthesis of new fluorescent tagged anionic porphyrins is currently in progress in our laboratory to resolve this problem.
Acknowledgements We thank the French National Research Agency (ANR PorphyPlant) for financial support.
Notes and references 1 V. Sarrazy, G. Garcia, J.-P. MBakidi, C. Le Morvan, G. Bégaud-Grimaud, R. Granet, V. Sol and P. Krausz,
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