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In vitro human cell responses to a low-dose photodynamic treatment vs. mild H2O2 exposure
Journal of Photochemistry and Photobiology B: Biology 143 (2015) 12–19
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In vitro human cell responses to a low-dose photodynamic treatment vs. mild H2O2 exposure Alfonso Blázquez-Castro ⇑, Juan C. Stockert Department of Biology, Autonomous University of Madrid, Madrid, Spain
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Article history: Received 25 September 2014 Received in revised form 7 December 2014 Accepted 13 December 2014 Available online 27 December 2014
a b s t r a c t Photodynamic treatments allow control of the amount of reactive oxygen species (ROS) produced through the photosensitizer concentration and the light dose delivered to the target. In this way low ROS doses can be achieved in situ to study cell responses related to redox regulation. In this study a comparison has been made between different cell responses to a low-dose photodynamic treatment and both low and relatively high concentrations of H2O2 in human immortalized keratinocytes (HaCaT). The obtained results show that the photodynamic treatment induces a stimulating cell response roughly equivalent to that produced by exposing cells to 10 5 M H2O2. Higher H2O2 concentrations gave rise to concentration-dependent deleterious effects on the cell cultures. Of importance is that the photodynamic treatment did not produce genotoxic damage, as measured by micronuclei frequency, while cultures exposed to 10 5 M H2O2 displayed a significant increase in the amount of cells with micronuclei. In summary, the low-dose photodynamic treatment promotes cell proliferation but does not incur in the excessive clastogenic lesions observed after H2O2 exposure. It is therefore proposed as a promising alternative to direct H2O2 exposure in the study of cell redox signalling. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The physiological function and signalling role of some Reactive oxygen species (ROS) is currently being accepted in mainstream Cell Biology [1–6]. Starting more than 20 years ago, experimental evidence has demonstrated that some ROS are produced by the cell machinery in order to signal for a whole range of responses [7–12]. This response range covers from cell proliferation [13–18] to programmed cell death [6,19–21] in opposing extremes. Between them, a manifold of different processes are controlled by the production of ROS, including, but not restricted to, cell differentiation [22,23], cytoskeletal control and cell migration [4,12], cell cycle control and arrest [21,24,25], senescence induction [6,8,19], inflammation [1,26], and wound healing [5,12,27,28]. This large range of cellular functions controlled/regulated by ROS points to their importance as one of the master nodes in the cell signalling pathways networks [21]. The physiological mechanism of action of ROS depends on the fine-tuned modulation of the intracellular and extracellular redox state (Ered). This Ered is an electrochemical value that depends directly on the concentrations of different molecules capable of ⇑ Corresponding author at: Aarhus Institute of Advanced Studies/Department of Chemistry, Aarhus University, Høegh-Guldbergs Gade 6B, DK-8000 Aarhus C, Denmark. Tel.: +45 87 15 53 45; fax: +45 86 19 61 99. E-mail address: [email protected] (A. Blázquez-Castro). http://dx.doi.org/10.1016/j.jphotobiol.2014.12.015 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
undergoing redox chemistry (i.e., exchange of electrons). The value of Ered can be found by using the Nernst equation of electromotive potential [3,29]. By definition ROS are molecules with oxidizing properties. They make the Ered value less negative. Meanwhile most organic compounds are reducing, changing Ered to more negative values. Several molecules act as reducing buffers in the cell, but the main compound is glutathione (GSH). Different cell responses depend directly on the Ered value. This is because many sulphur-containing biomolecules behave in different ways depending on the cell redox state by changing their chemical reactivity [30]. One of the most studied examples of this redox state regulation in cell signalling is the protein tyrosine phosphatases (PTPs). These proteins depend for their dephosphorylating function on a critical cysteine residue inside the catalytic pocket [31,32]. Above a certain concentration of ROS, or said in another way, below a certain Ered value this cysteine sulfhydryl residue is oxidized to a sulphenic state. This oxidation inactivates the catalytic pocket, leaving the PTP unable to carry out dephosphorylation. However, this oxidation is reversible in the presence of reducing compounds (e.g., GSH), which restore the enzymatic activity. Several molecules are included in the ROS chemical group, although not all of them are physiologically generated by cells as part of their signalling network. To play a role as a signalling agent a molecule must display several features like moderate reactivity, diffusivity and regulated production and scavenging mechanisms.
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Among ROS, the superoxide anion (O2 ) and hydrogen peroxide (H2O2) are the molecules which better fill the role of cellular messengers [1,2,33]. Given that O2 produces H2O2 by its dismutation, these chemical couple is employed thoroughly by all organisms to signal. In fact, H2O2 is the molecule of election for use in experiments to assess the role of ROS in cellular biology [34,35]. Recently, nevertheless, the possibility to use the photodynamic effect (PE) to produce small amounts of ROS to modulate cell behaviour, in counter point to cell killing, has started to provide a versatile methodology to study physiological responses [36– 39]. The possibility to induce, not only a non-damaging, but a genuine physiological response using a low-dose PE is a goal almost at grasp. With this previous research on mind, both the H2O2-related and the very recent PE achievements, we decided to compare the different responses in vitro of a human cell line (HaCaT) after H2O2 incubation vs. a low-dose photodynamic treatment. The underlying drive is to ascertain the similarities and differences between a photodynamic treatment and H2O2 exposure on cell cultures in order to take advantage of each methodology.
2. Material and methods
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Immediately after the PDT treatment, cell cultures were supplied with fresh complete medium before returning them to the cell incubator. During all procedures extreme care was taken to expose cell cultures to as less light as possible to avoid unwanted photodynamic side effects. It is worth mentioning that previous published results showed that either incubation with MAL for the established time or light exposure alone did not induce any ROS production under our PDT protocol [38].
2.3. Cell survival/growth assessment The survival/growth of cell cultures was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) assay. This assay has been demonstrated to be useful for measuring, not only cell survival, but also cell growth as a normalized value compared with a control [38]. Between 24 h and 72 h after ROS treatments cell cultures were incubated for 2 h with a solution of MTT (50 lg/mL) in DMEM at 37 °C. After this cell cultures were rinsed with PBS and 100 lL of DMSO (Panreac) was added to solubilize the produced formazan product. The optical density at 542 nm of the different cell wells was measured with a SpectraFluor plate reader (Tecan, Switzerland).
2.1. Cell culture The human immortalized keratinocyte cell line HaCaT was employed in these experiments. The cell cultures were grown in a complete medium, composed of Dulbecco´s Modified Eagle´s Medium (DMEM, Gibco, UK) supplemented with 10% foetal bovine serum (FBS, Gibco) and 0.5% antibiotics (penicillin G [10,000 U/ mL] and streptomycin sulphate [10,000 lg/mL], Gibco). Cell cultures were seeded at around 20–30% cell density and the experiments, unless otherwise indicated, were carried out when a 40– 60% cell density was achieved (exponential growth phase). Cells were grown and kept after experiments in a cell incubator that provided an atmosphere of 5% CO2, with a relative humidity of 95%, and a temperature of 37 °C (HERAcell, Kendro, Germany). When required for experiments cells were cultured on plastic multiwell plates (Costar, USA), Petri dishes (Corning, USA) or sterilized glass coverslips (Menzel-Gläser, Germany).
2.2. ROS treatments Cell cultures were exposed either to different H2O2 concentrations or to a low-dose PDT treatment. Exposure to H2O2 was achieved by diluting a stock solution of H2O2 (30% v/v, Panreac, Spain) in phosphate buffered saline (PBS, Gibco). Working peroxide solutions in the range 10 6 to 10 3 M were obtained. Cells were incubated with these solutions for 10 min, rinsed with fresh PBS, incubated in fresh complete medium and returned to the cell incubator. To induce a low-dose PDT treatment cell cultures were first incubated with the PpIX precursor methyl 5-aminolevulinate hydrochloride (MAL, Sigma, USA). Stock solutions of MAL were prepared in distilled water at 10 mM concentration. Cell cultures were incubated with a MAL solution (final concentration 0.1 mM) in DMEM for a total time of 5 h. Cultures were then rinsed in fresh PBS and exposed to red light for 10 min in PBS. The light source was a light emitting diode (LED) array (WP7143 SURC/E, Kingbright, USA) composed of 384 individual emitters. The peak emission was at 634 nm with a bandwidth at half-maximum of 17 nm, as measured with an UV–VIS–IR spectrometer (Fiberoptic, Avantes, Netherlands). The light source power density was 6.2 mW cm 2 at the cell culture irradiating position, measured with a photodiode (PAR 190 Li-1000, Li-Cor, USA).
2.4. Morphological cell death evaluation Cell cultures were evaluated for cell death attending to morphological criteria [40]. Between 24 h and 72 h after experimental treatments cell cultures were fixed with aqueous 3.7% formaldehyde (Panreac) for 20 min. Cells were rinsed with PBS, air dried and mounted with DePeX (Serva, USA). Microscopic inspection and counting was done under phase contrast optics (Olympus BX-61, Germany). At least 500 cells were morphologically evaluated for each treatment and time delay to obtain the results.
2.5. Mitotic index evaluation The mitotic index (MI) is defined here as the percentage of cells undergoing mitosis as compared with the total number of cells. Interphase and mitosis were allocated attending to nuclear morphological criteria. The MI was measured 24 h to 72 h after experimental procedures. Cell cultures were rinsed with fresh PBS and fixed with cold methanol ( 20 °C) for 5 min. Then they were stained for 5 min with a Hoechst 33258 (Sigma, USA) aqueous solution (2 lg/mL), washed with water, air dried and mounted in DePeX. The cell samples were evaluated under fluorescence microscopy (UV excitation: 365 nm; Olympus BX-61). No less than 1000 cells were assessed for each experimental condition at every time point.
2.6. Micronuclei genotoxicity evaluation The micronuclei assessment test is an adequate technique to evaluate clastogenic damage in cultured cells [41,42]. Usually the micronuclei formation is enhanced by an artificial procedure employing chemicals disrupting the microtubule machinery. However, the technique is also amenable to study spontaneous micronuclei [43]. In brief the technique employs the same protocol already described in Section 2.5 for MI measurement. Under UV excitation the number of micronuclei present surrounding the cell nucleus are counted after Hoechst 33258 staining and sample mounting. No less than 1000 cells were assessed for each experimental condition at every time point.
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2.7. Statistical analysis Data statistical analysis was done with the SPSS 15.0.1 statistical program (SPSS Inc, USA). The analysis of variance test – ANOVA – was the technique employed to evaluate the data. The post hoc Bonferroni test was used to determine between which experimental groups there were significant differences. 3. Results 3.1. Cell survival and growth assessment The MTT assay results obtained after ROS exposure (either PDT or H2O2 incubation) are shown in Fig. 1. Exposure to increasing concentrations of H2O2 first enhances cell growth (10 6–10 5 M) to then switch, at concentrations 10 4 and 10 3 M, to a negative impact on cell proliferation and survival at 24 h (Fig. 1A; see 3.2 below). The biphasic cell response is clearer at 48 h and 72 h after the treatments (Fig. 1B and C). The low-dose PDT treatment increases the cell population at all times measured as compared to control cultures, although it is no longer significant after 72 h. It is readily seen that the two treatment conditions showing a closer cell modulation effect are the low-dose PDT and incubation with 10 5 M H2O2. Incubation with this H2O2 concentration provides a significant cell increase as compared to the PDT treatment at 48 h (Fig. 1B). Incubation with 10 4 and 10 3 M H2O2 translates
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The results obtained with the MTT assay moved us to investigate the long-term cell fate after the different experimental conditions, in special to address the cell population decrease after 10 4 and 10 3 M H2O2 incubation. We proceeded to assess cell viability by attending to a morphological criterion, evaluating cells as viable, apoptotic or necrotic. The obtained results are presented in Fig. 2. The graphs show the frequency of viable (grey scale and striped bars), apoptotic (checkerboard pattern for all conditions) and necrotic (black bars for all conditions) as compared to the total cell population within a given experimental condition. An apoptotic minority population is observed in all cultures at 24 h. Nevertheless, this percentage increases significantly for cultures that had been exposed to 10 4 and 10 3 M H2O2 when compared with the control. These two experimental groups display some numbers of necrotic cells too. This trend continues at 48 h and 72 h for these two experimental conditions although both apoptotic and necrotic
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into ever decreasing cell population levels as time progresses. By 72 h cell cultures exposed to 10 3 M H2O2 show only 60% population numbers of the control culture.
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Fig. 1. Cell populations after the different ROS treatments as measured with the MTT colorimetric assay. The assessment took place at (A) 24 h, (B) 48 h and (C) 72 h after the experimental treatments. The concentrations refer to the H2O2 dilutions in PBS employed in each case. The data are expressed as average percentage of the corresponding control populations at each time. Bars denote one standard deviation. Symbols represent statistical significant differences: ⁄ p 6 0.05 compared to control; ⁄⁄ p 6 0.01 compared to control; § p 6 0.05 comparison between 10 5 M H2O2 and low-dose PDT treatments.
Fig. 2. Cell viability evaluation attending to morphological criteria. Cell viability categories are: viable (white, grey scale and stripped bars), apoptotic (checkerboard pattern bars) or necrotic (black bars) within each experimental condition. Viability was evaluated at (A) 24 h, (B) 48 h and (C) 72 h after experimental treatments. The concentrations refer to the H2O2 dilutions in PBS employed in each case. Data expressed as average percentage of cells in each category ± one standard deviation (at least 500 cells were evaluated in each experimental condition and time period). Statistical significance: ⁄ p 6 0.05 and ⁄⁄ p 6 0.01 compared to control viable frequency; § p 6 0.05 and §§ p 6 0.01 compared to control apoptotic frequency; – p 6 0.05 and –– p 6 0.01 compared to control necrotic frequency.
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percentages steadily decrease pointing to a population recovery (Fig. 2B and C). On the other side, regarding the milder ROS exposures with 10 6 and 10 5 M H2O2 and the PDT treatment, we found no significant differences between these and the control cultures. Cell viability was assessed between 95% and 100% at all measured times, with a small and ever decreasing apoptotic population the farther away from the ROS exposure event. This highlights the subtlety of the low concentration ROS treatments studied in this work.
The longer time frames (48 h and 72 h) showed no statistical differences between the control cultures and the different experimental treatments (Fig. 3B and C). Increased MI for low ROS conditions returned to control levels and high ROS conditions also recovered to a certain extent. However it deserves mention that the MI value of the PDT group at 48 h was on the verge of statistical significance (p = 0.096) as its average value clearly stands above the rest of experimental conditions (Fig. 3B). Some example images employed to evaluate MI from control and experimental cultures are shown in Fig. 3D. Cells undergoing mitosis (examples highlighted with arrows) show a flattened, sometimes ring-like, nuclear chromosomal arrangement and appear brighter on the fluorescence image. Interphase nuclei appear dimmer and tend to show a rounded or elliptic morphology.
3.3. Mitotic index measurement Given the diametrically opposite cell response induced by the low-ROS vs. high-ROS exposure already obtained, we analysed the MI as a complementary parameter that could provide information regarding either a stimulating or a deleterious event induced by ROS. Results are shown in Fig. 3. The MI measured at 24 h (Fig. 3A) provides further support to this biphasic trend. Cultures exposed to 10 5 M H2O2 showed a significant increase in their MI as compared to the control. So did cultures undergoing the lowdose PDT, which displayed even a more vigorous proliferation response. The opposite outcome was observed for the higher H2O2 concentration tested, where a significant proliferation inhibition took place.
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3.4. Micronuclei evaluation Finally the frequency of cells displaying micronuclei was analysed in order to get an estimate of genotoxic damage by the different experimental conditions. After 24 h all experimental treatments, except low dose PDT, showed an increase in micronuclei appearance (Fig. 4A). A statistical significant increase was found for cells that had been exposed to 10 4 and 10 3 M H2O2 for 24 h. At 48 h and 72 h after the experiments the amount of cells
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Fig. 3. Mitotic index assessment after different experimental conditions. The MI is represented as the percentage of cells in the culture undergoing mitosis (as evaluated by chromatin staining) at a given moment. The assessment took place (A) 24 h, (B) 48 h and (C) 72 h after the experimental treatments. The concentrations refer to the H2O2 dilutions in PBS employed in each case. Data presented as average percentage values in each category ± one standard deviation (at least 1000 cells were evaluated in each experimental condition and time period). Statistical significance: ⁄ p 6 0.05 and ⁄⁄ p 6 0.01 compared to control. (D) Examples of typical assessment HaCaT fluorescent images of Control, 10 6 M H2O2, 10 5 M H2O2 and PDT 24 h after the corresponding experimental treatment. Interphasic nuclei (dimmer rounded nuclei) can be readily distinguished from mitotic figures (brighter flattened nuclei highlighted with arrows). Bar: 25 lm.
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Micronuclei frequency (%)
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Fig. 4. Frequency of HaCaT cells displaying micronuclei after the experimental procedures at 24 h (A), 48 h (B) and 72 h (C). The concentrations refer to the H2O2 dilutions in PBS employed in each case. Results presented as average percentage values in each category ± one standard deviation (at least 1000 cells were evaluated in each experimental condition and time period). Note the change in scale between (A) and (B)–(C). Statistical significance: ⁄ p 6 0.05 and ⁄⁄ p 6 0.01 compared to control; § p 6 0.05 comparison between 10 5 M H2O2 and low-dose PDT treatments. (D) Examples of chromatin fluorescence images showing the typical morphology of normal nuclei and cells displaying micronuclei (arrows) for the indicated experimental conditions after 24 h. Bar: 10 lm.
displaying micronuclei increased drastically for some of the experimental treatments (please note the scale change in the micronuclei frequency at 48 h – Fig. 4B and 72 h – Fig. 4C). Previous exposure to 10 5, 10 4 and 10 3 M H2O2 increased significantly the amount of cells displaying micronuclei. Even exposure to 10 6 M H2O2 showed a maintained trend of higher micronuclei numbers as compared with the control, although this was not statistically significant. HaCaT cell cultures undergoing the low dose PDT showed the same amount of micronuclei as the controls at the three measured times (Fig. 4A–C). Comparing micronuclei frequency between low dose PDT and 10 5 M H2O2, the former condition consistently displayed a lower micronuclei frequency. This difference is statistically significant at 48 h and 72 h after the treatments. Some fluorescent HaCaT nuclei are shown in Fig. 4D, as examples of the image assessment employed to obtain the results shown in Fig. 4A–C. The images were taken 24 h after the corresponding treatment. Some nuclei display small rounded chromatin bodies, around 1 lm in diameter, detached from the nucleus. These micronuclei have been highlighted with arrows to help identification.
4. Discussion PDT is most usually associated with its clinical applications in Oncology and Dermatology. PDT on these areas goes back to more than four decades and it is a totally established clinical modality. However, the PE offers also the possibility to selectively produce
ROS in amounts that depend on the light dose provided to activate the process. Currently research on physiological signalling mediated by small ROS amounts and its impact on cell physiology is also becoming an operative field [1–4,35]. Scientific literature is starting to reflect this trend in which a low dose PDT treatment generates small but physiologically relevant amounts of ROS [36–39]. These engage a different cell response from the usual cell killing/ mitotic arrest responses so well established by now [44]. Under this new paradigm the most employed approach to the study of physiological ROS signalling is direct exposure to H2O2 [34,35]. The arguments behind this election are clear: H2O2 is easily handled and produced, and it is the most relevant ROS molecule, along with O2 , physiologically produced by cells to signal [1,12]. Currently it is an established fact that there is a H2O2 concentration gradient whereby moderate to high concentrations (10 4–10 3 M) induce cell death, cell cycle arrest and senescence in different in vitro and in vivo models [6,17,20,45], while low concentrations (10 7–10 5 M) modulate signalling pathways in order to enhance proliferation, wound healing, differentiation and tissue modelling [5,22,23,46]. In this line, Ibañez et al. had shown recently that scavenging the physiological levels of H2O2, both in vitro and in vivo, employing catalase leads to a cell cycle arrest at G1 through increased levels of p27KIP1 protein [47]. In light of our previous results of low dose PDT enhancement of proliferation in the HaCaT cell line [38], we found it pertinent to make a direct comparison with different H2O2 concentrations in order to ascertain the ‘‘equivalency’’ level of our PDT treatment. In general terms, the obtained results show that the low dose
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PDT was most similar in its overall effects to exposure to 10 5 M H2O2. Cell population measurements assessed by the MTT test show statistically significant increments in cell numbers for these two experimental conditions. However, exposure to 10 5 M H2O2 provided a stronger stimulating response in the long-term (48 h and 72 h). The whole range of data agree in trend with previous studies where low H2O2 concentrations increase cell numbers and increasing the amount of H2O2 leads to diminished populations [47–51]. Note, however, that we washed the peroxide solution after 10 min exposure to equalize its effects to the 10 min irradiation protocol for PDT. Most studies concerning the effects of H2O2 do not wash the peroxide solution. In their seminal paper Murrel et al. used different ways of producing ROS to assess human fibroblast responses [7]. One of them was providing H2O2 directly without further washing. They observed a significant increase in thymidine incorporation after cultures were exposed to 10 6 M H2O2. This is a lower figure than in our experiments but within an order of magnitude. Ohguro et al. followed this non-washing approach in their study of rabbit lens epithelial cells [48]. Nevertheless they obtained similar results as ours, although the stimulating concentration was again biased towards lower H2O2 concentrations (10 8–10 6 M). Perhaps these results reflect the different sensitivity to ROS of the cell cultures employed and/or the washing vs. non-washing protocol. Regarding cell viability assessed through morphology, our results show a subtle but significant reduction in viability for the highest H2O2 concentrations up to 72 h after treatments. As time progresses cell viability recovers for the 10 4 and 10 3 M H2O2 groups. Some necrotic cells were also observed, especially after 10 3 M H2O2 exposure, but the frequency was really low. The important point under the studied low dose conditions is that neither the lower concentrations of H2O2 (10 6 and 10 5 M) nor the PDT treatment showed significant differences in viability as compared to the control cultures. In contrast, Antunes and Cadenas found that very low H2O2 concentrations (5–50 lM) induced a very significant rise in apoptotic figures and also a non-negligible necrotic response [49]. Their experimental protocol, however, made use of a steady H2O2 biochemical production system based on glucose oxidation by a glucose oxidase. Their shortest exposure time was 30 min which represents an important difference as compared to our current experiments. Also they used a Jurkat T-cell culture which is a more sensible cell line than HaCaT. Our MI results come to support the results regarding cell population numbers and cell viability. Both 10 5 M H2O2 and PDT show a significant increase in the number of mitotic cells as compared to controls at 24 h post-treatment (Fig. 3A). The enhancement disappears at 48 h and 72 h. This punctual increase in the proliferation response explains the higher cell populations shown in Fig. 1 at all times for the two experimental treatments. However, because the MI reduces again to control levels at 48 h no further population increment is seen at 48 h and 72 h. Simply, the stimulated cell populations stay ahead of the control because of the initial mitotic signalling during the first 24 h. At the same time cultures exposed to 10 4 and 10 3 M H2O2 show a decrease in MI especially at 24 h. This partial mitotic arrest along with some cell death observed at 24 h and 48 h explain the observed significantly decreased cell populations at all measured times for these two high-ROS experimental conditions. The MI is a very sensitive target for assessing the biphasic cell response: either it is enhanced under cell cycle stimulating conditions or it is abolished under damaging ones to allow the cell to repair damage or undergo a programmed cell death [8,16,17,24,52]. This is exactly the observed trend in our experiments: a low dose ROS exposure increased temporally the MI
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above that of control (both for H2O2 and PDT), while a higher ROS concentration leads to cell damage and MI reduction. The differences observed in the amount of micronuclei induced by the low dose ROS treatments is an important result. The role of H2O2 in high concentrations as a clastogenic agent is well documented [41,42]. However, H2O2 seems to produce a high amount of micronuclei also at low concentrations. The present results show that the low dose PDT treatment induces a stimulating cell response that is, for all the assays studied, almost equal to exposure to 10 5 M H2O2 (Fig. 1–3). In contrast, cells incubated with 10 5 M H2O2 displayed an increased amount of micronuclei as compared with both control cultures and those exposed to low dose PDT. These differences became significant at 48 h and 72 h. Taken together this makes, from our point of view, a very important difference between the two types of treatments. Although both provide a way to transiently increase the proliferation rate in a cell culture, exposure to low concentration H2O2 increases dramatically the genomic instability. Even worse, results measuring cell death indicated that this micronuclei increase does not activate a cell death program even at 72 h after incubation (Fig. 2). In clear contrast, low dose PDT results in the same order of cell stimulation but with a safer margin regarding genomic preservation. One of the reasons to explain this difference between low concentration H2O2 and low dose PDT can be the lifetimes of the ROS involved in each case. The main ROS produced during PDT is singlet oxygen (1O2). Once produced 1O2 shows a very short lifetime (microseconds in aqueous medium). The main de-excitation pathway in an aqueous environment is vibrational relaxation and heat production [53]. Other relevant de-excitation pathway is chemical reaction with suitable cellular substrates [54]. The end result is that 1 O2 is consumed at the same rate that it is produced under biological conditions. So its diffusion distance, directly linked to its lifetime [53], is very short, usually cited much less than a micron. In contrast, H2O2 is a much more stable molecule. Its lifetime is measured in minutes and hours and it has plenty of time to diffuse across the cell [55]. This differences in lifetimes coupled to the different production site can explain the obtained results. With our methodology 1O2 is produced inside the cell (mitochondria and cytoplasm) while H2O2 is being provided from the outside where it diffuses into the different cell compartments. Even after washing, some H2O2 will remain inside the cell and keep diffusing. H2O2 can diffuse from the outside to the cell nucleus by traversing part of the cytoplasm. Then it can give rise to Fenton chemistry in the presence of transition metals (Fe2+/3+, Cu1+/2+) [49]. The outcome of Fenton chemistry is hydroxyl radical generation capable of producing chromatin lesions and, eventually, an increase in micronuclei frequency [41]. On the other hand, given the photosensitized production site of 1O2 and its short lifetime, it seems very improbable that it can reach the nucleus. However, both 1O2 and H2O2 can perturb the cell signalling pathways at the cytoplasm and plasmatic membrane in order to enhance cell proliferation. Although many of the results and proposed mechanisms presented in this work may apply in the field of low level laser (light) therapy (LLLT), we have restricted ourselves purposefully to the classical PDT scheme. In this scheme an exogenous PS (or physiological precursor in our case) is provided to mediate the energy transfer between light and ROS production. Nevertheless, it is clear from our point of view that the same biological principles guide both approaches, low dose PDT and LLLT, in their induction of physiological responses from the cell to the organism levels. This is exemplified in previous works in LLLT such as, for example, that of Lavi et al. where very similar H2O2 concentrations (12–48 lM) to those examined by us were compared to LLLT effect with visible
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light in regard to intracellular Ca2+ levels, cell viability and cell damage in rat cardiomyocytes [56]. 5. Conclusion The results presented indicate that a low dose PDT treatment promotes an increase in cell proliferation and that this cell response is almost equal to that obtained by exposing cell cultures to 10 5 M H2O2 for a short time (10 min). However, the PDT treatment showed no increase in micronuclei frequency while 10 5 M H2O2 gave rise to a statistically significant amount of micronuclei. Accordingly, we propose the use of low dose PDT treatments as a less genotoxic alternative for the study of physiological cell signalling mediated by low ROS concentrations. 6. Abbreviations
GSH H2O2 LLLT MAL MI MTT 1
O2 O2 PDT PE PS ROS
glutathione hydrogen peroxide low level laser (light) therapy methyl 5-aminolevulinate hydrochloride mitotic index 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide singlet oxygen (a1Dg) superoxide anion photodynamic therapy photodynamic effect photosensitizer reactive oxygen species
Acknowledgements The authors acknowledge Antonio Romero and Dr. Begoña López-Arias for fruitful discussions and experimental planning. ABC acknowledges financial support from Ministerio de Ciencia e Innovación, Spain through a FPU Fellowship. References [1] W.A. Pryor, K.N. Houk, C.S. Foote, J.M. Fukuto, L.J. Ignarro, G.L. Squadrito, K.J.A. Davies, Free radical biology and medicine: it’s a gas, man!, Am J. Physiol. Regul. Integr. Comp. Physiol. 291 (2006) R491–R511. [2] B. D’Autréaux, M.B. Toledano, ROS as signaling molecules: mechanisms that generate specificity in ROS homeostasis, Nat. Rev. 8 (2007) 813–824. [3] D.P. Jones, Radical-free biology of oxidative stress, Am. J. Physiol. Cell Physiol. 295 (2008) C849–C868. [4] F. Jiang, Y. Zhang, G.J. Dusting, NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair, Pharmacol. Rev. 63 (2011) 218–242. [5] N.R. Love, Y. Chen, S. Ishibashi, P. Kritsiligkou, R. Lea, Y. Koh, J.L. Gallop, K. Dorey, E. Amaya, Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration, Nat. Cell Biol. 15 (2013) 222–228. [6] E. Panieri, V. Gogvadze, E. Norberg, R. Venkatesh, S. Orrenius, B. Zhivotovsky, Reactive oxygen species generated in different compartments induce cell death, survival, or senescence, Free Radical Biol. Med. 57 (2013) 176–187. [7] G.A.C. Murrel, M.J.O. Francis, L. Bromley, Modulation of fibroblast proliferation by oxygen free radicals, Biochem. J. 265 (1990) 659–665. [8] J. Boonstra, J.A. Post, Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells, Gene 337 (2004) 1–13. [9] Y.M. Go, D.P. Jones, Redox compartmentalization in eukaryotic cells, Biochim. Biophys. Acta 1780 (2008) 1273–1290. [10] D.P. Jones, Y.M. Go, Redox compartmentalization and cellular stress, Diabetes Obesity Metab. 12 (2010) 116–125. [11] G.R. Buettner, B.A. Wagner, V.G.J. Rodgers, Quantitative redox biology: an approach to understand the role of reactive species in defining the cellular redox environment, Cell Biochem. Biophys. 67 (2013) 477–483.
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Chandel, Mitochondrial reactive oxygen species promote epidermal differentiation and hair follicle development, Sci. Signal. 6 (2013) 1–9. [24] S. Kurata, Selective activation of p38 MAPK cascade and mitotic arrest caused by low level oxidative stress, J. Biol. Chem. 275 (2000) 23413–23416. [25] M. Li, L. Zhao, J. Liu, A.L. Liu, W.S. Zeng, S.Q. Luo, X.C. Bai, Hydrogen peroxide induces G2 cell cycle arrest and inhibits cell proliferation in osteoblasts, Anat. Rec. 292 (2009) 1107–1113. [26] T. Finkel, Signal transduction by mitochondrial oxidants, J. Biol. Chem. 287 (2012) 4434–4440. [27] C.K. Sen, The general case for redox control of wound repair, Wound Repair Regen. 11 (2003) 431–438. [28] C.K. Sen, S. Roy, Redox signals in wound healing, Biochim. Biophys. Acta 1780 (2008) 1348–1361. [29] V. Mallikarjun, D.J. Clarke, C.J. Campbell, Cellular redox potential and the biomolecular electrochemical series: a systems hypothesis, Free Radical Biol. Med. 53 (2012) 280–288. [30] Y.M. Go, D.P. Jones, Thiol/disulphide redox states in signaling and sensing, Crit. Rev. Biochem. Mol. Biol. 48 (2013) 173–191. [31] K. Lee, W.J. Esselman, Inhibition of PTPs by H2O2 regulates the activation of distinct MAPK pathways, Free Radical Biol. Med. 33 (2002) 1121–1132. [32] P. Chiarugi, P. Chirri, Redox regulation of protein tyrosine phosphatases during receptor tyrosine kinase signal transduction, Trends Biochem. Sci. 28 (2003) 509–514. [33] R.M. Day, Y.J. Suzuki, Cell proliferation, reactive oxygen and cellular glutathione, Dose Response 3 (2005) 425–442. [34] E.A. Veal, A.M. Day, B.A. Morgan, Hydrogen peroxide sensing and signaling, Mol. Cell 26 (2007) 1–14. [35] D.R. Gough, T.G. Cotter, Hydrogen peroxide: a Jekyll and Hyde signalling molecule, Cell Death Dis. 2 (2011) e213. [36] X. Zhang, F. Jiang, Z. Gang Zhang, S.N. Kalkanis, X. Hong, A.C. deCarvalho, J. Chen, H. Yang, A.M. Robin, M. Chopp, Low-dose photodynamic therapy increases endothelial cell proliferation and VEGF expression in nude mice brain, Lasers Med. Sci. 20 (2005) 74–79. [37] O. Bozkulak, S. Wong, M. Luna, A. Ferrario, N. Rucker, M. Gulsoy, C.J. Gomer, Multiple components of photodynamic therapy can phosphorylate Akt, Photochem. Photobiol. 83 (2007) 1029–1033. [38] A. Blázquez-Castro, E. Carrasco, M.I. Calvo, P. Jaén, J.C. Stockert, A. Juarranz, F. Sanz-Rodríguez, J. Espada, Protoporphyrin IX-dependent photodynamic production of endogenous ROS stimulates cell proliferation, Eur. J. Cell Biol. 91 (2012) 216–223. [39] A. Blázquez-Castro, T. Breitenbach, P.R. Ogilby, Singlet oxygen and ROS in a new light: low-dose subcellular photodynamic treatment enhances proliferation at the single cell level, Photochem. Photobiol. Sci. 13 (2014) 1235–1240. [40] A. Gollmer, F. Besostri, T. Breitenbach, P.R. Ogilby, Spatially resolved twophoton irradiation of an intracellular singlet oxygen photosensitizer: correlating cell response to the site of localized irradiation, Free Radical Res. 47 (2013) 718–730. [41] M. Fenech, J. Crott, J. Turner, S. Brown, Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide, Mutagenesis 14 (1999) 605–612. [42] R. Mateuca, N. Lombaert, P.V. Aka, I. Decordier, M. Kirsch-Volders, Chromosomal changes: induction, detection methods and applicability in human biomonitoring, Biochimie 88 (2006) 1515–1531. [43] X. Rao, Y. Zhang, Q. Yi, H. Hou, B. Xu, L. Chu, Y. Huang, W. Zhang, M. Fenech, Q. Shi, Multiple origins of spontaneously arising micronuclei in HeLa cells: direct evidence from long-term live cell imaging, Mutat. Res. 646 (2008) 41–49.
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
In vitro human cell responses to a low-dose photodynamic treatment vs. mild H2O2 exposure Alfonso Blázquez-Castro ⇑, Juan C. Stockert Department of Biology, Autonomous University of Madrid, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 25 September 2014 Received in revised form 7 December 2014 Accepted 13 December 2014 Available online 27 December 2014
a b s t r a c t Photodynamic treatments allow control of the amount of reactive oxygen species (ROS) produced through the photosensitizer concentration and the light dose delivered to the target. In this way low ROS doses can be achieved in situ to study cell responses related to redox regulation. In this study a comparison has been made between different cell responses to a low-dose photodynamic treatment and both low and relatively high concentrations of H2O2 in human immortalized keratinocytes (HaCaT). The obtained results show that the photodynamic treatment induces a stimulating cell response roughly equivalent to that produced by exposing cells to 10 5 M H2O2. Higher H2O2 concentrations gave rise to concentration-dependent deleterious effects on the cell cultures. Of importance is that the photodynamic treatment did not produce genotoxic damage, as measured by micronuclei frequency, while cultures exposed to 10 5 M H2O2 displayed a significant increase in the amount of cells with micronuclei. In summary, the low-dose photodynamic treatment promotes cell proliferation but does not incur in the excessive clastogenic lesions observed after H2O2 exposure. It is therefore proposed as a promising alternative to direct H2O2 exposure in the study of cell redox signalling. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The physiological function and signalling role of some Reactive oxygen species (ROS) is currently being accepted in mainstream Cell Biology [1–6]. Starting more than 20 years ago, experimental evidence has demonstrated that some ROS are produced by the cell machinery in order to signal for a whole range of responses [7–12]. This response range covers from cell proliferation [13–18] to programmed cell death [6,19–21] in opposing extremes. Between them, a manifold of different processes are controlled by the production of ROS, including, but not restricted to, cell differentiation [22,23], cytoskeletal control and cell migration [4,12], cell cycle control and arrest [21,24,25], senescence induction [6,8,19], inflammation [1,26], and wound healing [5,12,27,28]. This large range of cellular functions controlled/regulated by ROS points to their importance as one of the master nodes in the cell signalling pathways networks [21]. The physiological mechanism of action of ROS depends on the fine-tuned modulation of the intracellular and extracellular redox state (Ered). This Ered is an electrochemical value that depends directly on the concentrations of different molecules capable of ⇑ Corresponding author at: Aarhus Institute of Advanced Studies/Department of Chemistry, Aarhus University, Høegh-Guldbergs Gade 6B, DK-8000 Aarhus C, Denmark. Tel.: +45 87 15 53 45; fax: +45 86 19 61 99. E-mail address: [email protected] (A. Blázquez-Castro). http://dx.doi.org/10.1016/j.jphotobiol.2014.12.015 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
undergoing redox chemistry (i.e., exchange of electrons). The value of Ered can be found by using the Nernst equation of electromotive potential [3,29]. By definition ROS are molecules with oxidizing properties. They make the Ered value less negative. Meanwhile most organic compounds are reducing, changing Ered to more negative values. Several molecules act as reducing buffers in the cell, but the main compound is glutathione (GSH). Different cell responses depend directly on the Ered value. This is because many sulphur-containing biomolecules behave in different ways depending on the cell redox state by changing their chemical reactivity [30]. One of the most studied examples of this redox state regulation in cell signalling is the protein tyrosine phosphatases (PTPs). These proteins depend for their dephosphorylating function on a critical cysteine residue inside the catalytic pocket [31,32]. Above a certain concentration of ROS, or said in another way, below a certain Ered value this cysteine sulfhydryl residue is oxidized to a sulphenic state. This oxidation inactivates the catalytic pocket, leaving the PTP unable to carry out dephosphorylation. However, this oxidation is reversible in the presence of reducing compounds (e.g., GSH), which restore the enzymatic activity. Several molecules are included in the ROS chemical group, although not all of them are physiologically generated by cells as part of their signalling network. To play a role as a signalling agent a molecule must display several features like moderate reactivity, diffusivity and regulated production and scavenging mechanisms.
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Among ROS, the superoxide anion (O2 ) and hydrogen peroxide (H2O2) are the molecules which better fill the role of cellular messengers [1,2,33]. Given that O2 produces H2O2 by its dismutation, these chemical couple is employed thoroughly by all organisms to signal. In fact, H2O2 is the molecule of election for use in experiments to assess the role of ROS in cellular biology [34,35]. Recently, nevertheless, the possibility to use the photodynamic effect (PE) to produce small amounts of ROS to modulate cell behaviour, in counter point to cell killing, has started to provide a versatile methodology to study physiological responses [36– 39]. The possibility to induce, not only a non-damaging, but a genuine physiological response using a low-dose PE is a goal almost at grasp. With this previous research on mind, both the H2O2-related and the very recent PE achievements, we decided to compare the different responses in vitro of a human cell line (HaCaT) after H2O2 incubation vs. a low-dose photodynamic treatment. The underlying drive is to ascertain the similarities and differences between a photodynamic treatment and H2O2 exposure on cell cultures in order to take advantage of each methodology.
2. Material and methods
13
Immediately after the PDT treatment, cell cultures were supplied with fresh complete medium before returning them to the cell incubator. During all procedures extreme care was taken to expose cell cultures to as less light as possible to avoid unwanted photodynamic side effects. It is worth mentioning that previous published results showed that either incubation with MAL for the established time or light exposure alone did not induce any ROS production under our PDT protocol [38].
2.3. Cell survival/growth assessment The survival/growth of cell cultures was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) assay. This assay has been demonstrated to be useful for measuring, not only cell survival, but also cell growth as a normalized value compared with a control [38]. Between 24 h and 72 h after ROS treatments cell cultures were incubated for 2 h with a solution of MTT (50 lg/mL) in DMEM at 37 °C. After this cell cultures were rinsed with PBS and 100 lL of DMSO (Panreac) was added to solubilize the produced formazan product. The optical density at 542 nm of the different cell wells was measured with a SpectraFluor plate reader (Tecan, Switzerland).
2.1. Cell culture The human immortalized keratinocyte cell line HaCaT was employed in these experiments. The cell cultures were grown in a complete medium, composed of Dulbecco´s Modified Eagle´s Medium (DMEM, Gibco, UK) supplemented with 10% foetal bovine serum (FBS, Gibco) and 0.5% antibiotics (penicillin G [10,000 U/ mL] and streptomycin sulphate [10,000 lg/mL], Gibco). Cell cultures were seeded at around 20–30% cell density and the experiments, unless otherwise indicated, were carried out when a 40– 60% cell density was achieved (exponential growth phase). Cells were grown and kept after experiments in a cell incubator that provided an atmosphere of 5% CO2, with a relative humidity of 95%, and a temperature of 37 °C (HERAcell, Kendro, Germany). When required for experiments cells were cultured on plastic multiwell plates (Costar, USA), Petri dishes (Corning, USA) or sterilized glass coverslips (Menzel-Gläser, Germany).
2.2. ROS treatments Cell cultures were exposed either to different H2O2 concentrations or to a low-dose PDT treatment. Exposure to H2O2 was achieved by diluting a stock solution of H2O2 (30% v/v, Panreac, Spain) in phosphate buffered saline (PBS, Gibco). Working peroxide solutions in the range 10 6 to 10 3 M were obtained. Cells were incubated with these solutions for 10 min, rinsed with fresh PBS, incubated in fresh complete medium and returned to the cell incubator. To induce a low-dose PDT treatment cell cultures were first incubated with the PpIX precursor methyl 5-aminolevulinate hydrochloride (MAL, Sigma, USA). Stock solutions of MAL were prepared in distilled water at 10 mM concentration. Cell cultures were incubated with a MAL solution (final concentration 0.1 mM) in DMEM for a total time of 5 h. Cultures were then rinsed in fresh PBS and exposed to red light for 10 min in PBS. The light source was a light emitting diode (LED) array (WP7143 SURC/E, Kingbright, USA) composed of 384 individual emitters. The peak emission was at 634 nm with a bandwidth at half-maximum of 17 nm, as measured with an UV–VIS–IR spectrometer (Fiberoptic, Avantes, Netherlands). The light source power density was 6.2 mW cm 2 at the cell culture irradiating position, measured with a photodiode (PAR 190 Li-1000, Li-Cor, USA).
2.4. Morphological cell death evaluation Cell cultures were evaluated for cell death attending to morphological criteria [40]. Between 24 h and 72 h after experimental treatments cell cultures were fixed with aqueous 3.7% formaldehyde (Panreac) for 20 min. Cells were rinsed with PBS, air dried and mounted with DePeX (Serva, USA). Microscopic inspection and counting was done under phase contrast optics (Olympus BX-61, Germany). At least 500 cells were morphologically evaluated for each treatment and time delay to obtain the results.
2.5. Mitotic index evaluation The mitotic index (MI) is defined here as the percentage of cells undergoing mitosis as compared with the total number of cells. Interphase and mitosis were allocated attending to nuclear morphological criteria. The MI was measured 24 h to 72 h after experimental procedures. Cell cultures were rinsed with fresh PBS and fixed with cold methanol ( 20 °C) for 5 min. Then they were stained for 5 min with a Hoechst 33258 (Sigma, USA) aqueous solution (2 lg/mL), washed with water, air dried and mounted in DePeX. The cell samples were evaluated under fluorescence microscopy (UV excitation: 365 nm; Olympus BX-61). No less than 1000 cells were assessed for each experimental condition at every time point.
2.6. Micronuclei genotoxicity evaluation The micronuclei assessment test is an adequate technique to evaluate clastogenic damage in cultured cells [41,42]. Usually the micronuclei formation is enhanced by an artificial procedure employing chemicals disrupting the microtubule machinery. However, the technique is also amenable to study spontaneous micronuclei [43]. In brief the technique employs the same protocol already described in Section 2.5 for MI measurement. Under UV excitation the number of micronuclei present surrounding the cell nucleus are counted after Hoechst 33258 staining and sample mounting. No less than 1000 cells were assessed for each experimental condition at every time point.
A. Blázquez-Castro, J.C. Stockert / Journal of Photochemistry and Photobiology B: Biology 143 (2015) 12–19
2.7. Statistical analysis Data statistical analysis was done with the SPSS 15.0.1 statistical program (SPSS Inc, USA). The analysis of variance test – ANOVA – was the technique employed to evaluate the data. The post hoc Bonferroni test was used to determine between which experimental groups there were significant differences. 3. Results 3.1. Cell survival and growth assessment The MTT assay results obtained after ROS exposure (either PDT or H2O2 incubation) are shown in Fig. 1. Exposure to increasing concentrations of H2O2 first enhances cell growth (10 6–10 5 M) to then switch, at concentrations 10 4 and 10 3 M, to a negative impact on cell proliferation and survival at 24 h (Fig. 1A; see 3.2 below). The biphasic cell response is clearer at 48 h and 72 h after the treatments (Fig. 1B and C). The low-dose PDT treatment increases the cell population at all times measured as compared to control cultures, although it is no longer significant after 72 h. It is readily seen that the two treatment conditions showing a closer cell modulation effect are the low-dose PDT and incubation with 10 5 M H2O2. Incubation with this H2O2 concentration provides a significant cell increase as compared to the PDT treatment at 48 h (Fig. 1B). Incubation with 10 4 and 10 3 M H2O2 translates
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The results obtained with the MTT assay moved us to investigate the long-term cell fate after the different experimental conditions, in special to address the cell population decrease after 10 4 and 10 3 M H2O2 incubation. We proceeded to assess cell viability by attending to a morphological criterion, evaluating cells as viable, apoptotic or necrotic. The obtained results are presented in Fig. 2. The graphs show the frequency of viable (grey scale and striped bars), apoptotic (checkerboard pattern for all conditions) and necrotic (black bars for all conditions) as compared to the total cell population within a given experimental condition. An apoptotic minority population is observed in all cultures at 24 h. Nevertheless, this percentage increases significantly for cultures that had been exposed to 10 4 and 10 3 M H2O2 when compared with the control. These two experimental groups display some numbers of necrotic cells too. This trend continues at 48 h and 72 h for these two experimental conditions although both apoptotic and necrotic
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Fig. 1. Cell populations after the different ROS treatments as measured with the MTT colorimetric assay. The assessment took place at (A) 24 h, (B) 48 h and (C) 72 h after the experimental treatments. The concentrations refer to the H2O2 dilutions in PBS employed in each case. The data are expressed as average percentage of the corresponding control populations at each time. Bars denote one standard deviation. Symbols represent statistical significant differences: ⁄ p 6 0.05 compared to control; ⁄⁄ p 6 0.01 compared to control; § p 6 0.05 comparison between 10 5 M H2O2 and low-dose PDT treatments.
Fig. 2. Cell viability evaluation attending to morphological criteria. Cell viability categories are: viable (white, grey scale and stripped bars), apoptotic (checkerboard pattern bars) or necrotic (black bars) within each experimental condition. Viability was evaluated at (A) 24 h, (B) 48 h and (C) 72 h after experimental treatments. The concentrations refer to the H2O2 dilutions in PBS employed in each case. Data expressed as average percentage of cells in each category ± one standard deviation (at least 500 cells were evaluated in each experimental condition and time period). Statistical significance: ⁄ p 6 0.05 and ⁄⁄ p 6 0.01 compared to control viable frequency; § p 6 0.05 and §§ p 6 0.01 compared to control apoptotic frequency; – p 6 0.05 and –– p 6 0.01 compared to control necrotic frequency.
A. Blázquez-Castro, J.C. Stockert / Journal of Photochemistry and Photobiology B: Biology 143 (2015) 12–19
percentages steadily decrease pointing to a population recovery (Fig. 2B and C). On the other side, regarding the milder ROS exposures with 10 6 and 10 5 M H2O2 and the PDT treatment, we found no significant differences between these and the control cultures. Cell viability was assessed between 95% and 100% at all measured times, with a small and ever decreasing apoptotic population the farther away from the ROS exposure event. This highlights the subtlety of the low concentration ROS treatments studied in this work.
The longer time frames (48 h and 72 h) showed no statistical differences between the control cultures and the different experimental treatments (Fig. 3B and C). Increased MI for low ROS conditions returned to control levels and high ROS conditions also recovered to a certain extent. However it deserves mention that the MI value of the PDT group at 48 h was on the verge of statistical significance (p = 0.096) as its average value clearly stands above the rest of experimental conditions (Fig. 3B). Some example images employed to evaluate MI from control and experimental cultures are shown in Fig. 3D. Cells undergoing mitosis (examples highlighted with arrows) show a flattened, sometimes ring-like, nuclear chromosomal arrangement and appear brighter on the fluorescence image. Interphase nuclei appear dimmer and tend to show a rounded or elliptic morphology.
3.3. Mitotic index measurement Given the diametrically opposite cell response induced by the low-ROS vs. high-ROS exposure already obtained, we analysed the MI as a complementary parameter that could provide information regarding either a stimulating or a deleterious event induced by ROS. Results are shown in Fig. 3. The MI measured at 24 h (Fig. 3A) provides further support to this biphasic trend. Cultures exposed to 10 5 M H2O2 showed a significant increase in their MI as compared to the control. So did cultures undergoing the lowdose PDT, which displayed even a more vigorous proliferation response. The opposite outcome was observed for the higher H2O2 concentration tested, where a significant proliferation inhibition took place.
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3.4. Micronuclei evaluation Finally the frequency of cells displaying micronuclei was analysed in order to get an estimate of genotoxic damage by the different experimental conditions. After 24 h all experimental treatments, except low dose PDT, showed an increase in micronuclei appearance (Fig. 4A). A statistical significant increase was found for cells that had been exposed to 10 4 and 10 3 M H2O2 for 24 h. At 48 h and 72 h after the experiments the amount of cells
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Fig. 3. Mitotic index assessment after different experimental conditions. The MI is represented as the percentage of cells in the culture undergoing mitosis (as evaluated by chromatin staining) at a given moment. The assessment took place (A) 24 h, (B) 48 h and (C) 72 h after the experimental treatments. The concentrations refer to the H2O2 dilutions in PBS employed in each case. Data presented as average percentage values in each category ± one standard deviation (at least 1000 cells were evaluated in each experimental condition and time period). Statistical significance: ⁄ p 6 0.05 and ⁄⁄ p 6 0.01 compared to control. (D) Examples of typical assessment HaCaT fluorescent images of Control, 10 6 M H2O2, 10 5 M H2O2 and PDT 24 h after the corresponding experimental treatment. Interphasic nuclei (dimmer rounded nuclei) can be readily distinguished from mitotic figures (brighter flattened nuclei highlighted with arrows). Bar: 25 lm.
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Micronuclei frequency (%)
(A)
Micronuclei frequency (%)
(B)
Micronuclei frequency (%)
(C)
10
24 h
(D)
**
Control
**
8 6 4
**
2 0 25
48 h
H2O2 10-5 M
**
20
**
15
§
*
10 5 0 25
PDT
72 h
**
**
20 15 §
10
*
5 0
Control
10-6 M
10-5 M
10-4 M
10-3 M
PDT
Fig. 4. Frequency of HaCaT cells displaying micronuclei after the experimental procedures at 24 h (A), 48 h (B) and 72 h (C). The concentrations refer to the H2O2 dilutions in PBS employed in each case. Results presented as average percentage values in each category ± one standard deviation (at least 1000 cells were evaluated in each experimental condition and time period). Note the change in scale between (A) and (B)–(C). Statistical significance: ⁄ p 6 0.05 and ⁄⁄ p 6 0.01 compared to control; § p 6 0.05 comparison between 10 5 M H2O2 and low-dose PDT treatments. (D) Examples of chromatin fluorescence images showing the typical morphology of normal nuclei and cells displaying micronuclei (arrows) for the indicated experimental conditions after 24 h. Bar: 10 lm.
displaying micronuclei increased drastically for some of the experimental treatments (please note the scale change in the micronuclei frequency at 48 h – Fig. 4B and 72 h – Fig. 4C). Previous exposure to 10 5, 10 4 and 10 3 M H2O2 increased significantly the amount of cells displaying micronuclei. Even exposure to 10 6 M H2O2 showed a maintained trend of higher micronuclei numbers as compared with the control, although this was not statistically significant. HaCaT cell cultures undergoing the low dose PDT showed the same amount of micronuclei as the controls at the three measured times (Fig. 4A–C). Comparing micronuclei frequency between low dose PDT and 10 5 M H2O2, the former condition consistently displayed a lower micronuclei frequency. This difference is statistically significant at 48 h and 72 h after the treatments. Some fluorescent HaCaT nuclei are shown in Fig. 4D, as examples of the image assessment employed to obtain the results shown in Fig. 4A–C. The images were taken 24 h after the corresponding treatment. Some nuclei display small rounded chromatin bodies, around 1 lm in diameter, detached from the nucleus. These micronuclei have been highlighted with arrows to help identification.
4. Discussion PDT is most usually associated with its clinical applications in Oncology and Dermatology. PDT on these areas goes back to more than four decades and it is a totally established clinical modality. However, the PE offers also the possibility to selectively produce
ROS in amounts that depend on the light dose provided to activate the process. Currently research on physiological signalling mediated by small ROS amounts and its impact on cell physiology is also becoming an operative field [1–4,35]. Scientific literature is starting to reflect this trend in which a low dose PDT treatment generates small but physiologically relevant amounts of ROS [36–39]. These engage a different cell response from the usual cell killing/ mitotic arrest responses so well established by now [44]. Under this new paradigm the most employed approach to the study of physiological ROS signalling is direct exposure to H2O2 [34,35]. The arguments behind this election are clear: H2O2 is easily handled and produced, and it is the most relevant ROS molecule, along with O2 , physiologically produced by cells to signal [1,12]. Currently it is an established fact that there is a H2O2 concentration gradient whereby moderate to high concentrations (10 4–10 3 M) induce cell death, cell cycle arrest and senescence in different in vitro and in vivo models [6,17,20,45], while low concentrations (10 7–10 5 M) modulate signalling pathways in order to enhance proliferation, wound healing, differentiation and tissue modelling [5,22,23,46]. In this line, Ibañez et al. had shown recently that scavenging the physiological levels of H2O2, both in vitro and in vivo, employing catalase leads to a cell cycle arrest at G1 through increased levels of p27KIP1 protein [47]. In light of our previous results of low dose PDT enhancement of proliferation in the HaCaT cell line [38], we found it pertinent to make a direct comparison with different H2O2 concentrations in order to ascertain the ‘‘equivalency’’ level of our PDT treatment. In general terms, the obtained results show that the low dose
A. Blázquez-Castro, J.C. Stockert / Journal of Photochemistry and Photobiology B: Biology 143 (2015) 12–19
PDT was most similar in its overall effects to exposure to 10 5 M H2O2. Cell population measurements assessed by the MTT test show statistically significant increments in cell numbers for these two experimental conditions. However, exposure to 10 5 M H2O2 provided a stronger stimulating response in the long-term (48 h and 72 h). The whole range of data agree in trend with previous studies where low H2O2 concentrations increase cell numbers and increasing the amount of H2O2 leads to diminished populations [47–51]. Note, however, that we washed the peroxide solution after 10 min exposure to equalize its effects to the 10 min irradiation protocol for PDT. Most studies concerning the effects of H2O2 do not wash the peroxide solution. In their seminal paper Murrel et al. used different ways of producing ROS to assess human fibroblast responses [7]. One of them was providing H2O2 directly without further washing. They observed a significant increase in thymidine incorporation after cultures were exposed to 10 6 M H2O2. This is a lower figure than in our experiments but within an order of magnitude. Ohguro et al. followed this non-washing approach in their study of rabbit lens epithelial cells [48]. Nevertheless they obtained similar results as ours, although the stimulating concentration was again biased towards lower H2O2 concentrations (10 8–10 6 M). Perhaps these results reflect the different sensitivity to ROS of the cell cultures employed and/or the washing vs. non-washing protocol. Regarding cell viability assessed through morphology, our results show a subtle but significant reduction in viability for the highest H2O2 concentrations up to 72 h after treatments. As time progresses cell viability recovers for the 10 4 and 10 3 M H2O2 groups. Some necrotic cells were also observed, especially after 10 3 M H2O2 exposure, but the frequency was really low. The important point under the studied low dose conditions is that neither the lower concentrations of H2O2 (10 6 and 10 5 M) nor the PDT treatment showed significant differences in viability as compared to the control cultures. In contrast, Antunes and Cadenas found that very low H2O2 concentrations (5–50 lM) induced a very significant rise in apoptotic figures and also a non-negligible necrotic response [49]. Their experimental protocol, however, made use of a steady H2O2 biochemical production system based on glucose oxidation by a glucose oxidase. Their shortest exposure time was 30 min which represents an important difference as compared to our current experiments. Also they used a Jurkat T-cell culture which is a more sensible cell line than HaCaT. Our MI results come to support the results regarding cell population numbers and cell viability. Both 10 5 M H2O2 and PDT show a significant increase in the number of mitotic cells as compared to controls at 24 h post-treatment (Fig. 3A). The enhancement disappears at 48 h and 72 h. This punctual increase in the proliferation response explains the higher cell populations shown in Fig. 1 at all times for the two experimental treatments. However, because the MI reduces again to control levels at 48 h no further population increment is seen at 48 h and 72 h. Simply, the stimulated cell populations stay ahead of the control because of the initial mitotic signalling during the first 24 h. At the same time cultures exposed to 10 4 and 10 3 M H2O2 show a decrease in MI especially at 24 h. This partial mitotic arrest along with some cell death observed at 24 h and 48 h explain the observed significantly decreased cell populations at all measured times for these two high-ROS experimental conditions. The MI is a very sensitive target for assessing the biphasic cell response: either it is enhanced under cell cycle stimulating conditions or it is abolished under damaging ones to allow the cell to repair damage or undergo a programmed cell death [8,16,17,24,52]. This is exactly the observed trend in our experiments: a low dose ROS exposure increased temporally the MI
17
above that of control (both for H2O2 and PDT), while a higher ROS concentration leads to cell damage and MI reduction. The differences observed in the amount of micronuclei induced by the low dose ROS treatments is an important result. The role of H2O2 in high concentrations as a clastogenic agent is well documented [41,42]. However, H2O2 seems to produce a high amount of micronuclei also at low concentrations. The present results show that the low dose PDT treatment induces a stimulating cell response that is, for all the assays studied, almost equal to exposure to 10 5 M H2O2 (Fig. 1–3). In contrast, cells incubated with 10 5 M H2O2 displayed an increased amount of micronuclei as compared with both control cultures and those exposed to low dose PDT. These differences became significant at 48 h and 72 h. Taken together this makes, from our point of view, a very important difference between the two types of treatments. Although both provide a way to transiently increase the proliferation rate in a cell culture, exposure to low concentration H2O2 increases dramatically the genomic instability. Even worse, results measuring cell death indicated that this micronuclei increase does not activate a cell death program even at 72 h after incubation (Fig. 2). In clear contrast, low dose PDT results in the same order of cell stimulation but with a safer margin regarding genomic preservation. One of the reasons to explain this difference between low concentration H2O2 and low dose PDT can be the lifetimes of the ROS involved in each case. The main ROS produced during PDT is singlet oxygen (1O2). Once produced 1O2 shows a very short lifetime (microseconds in aqueous medium). The main de-excitation pathway in an aqueous environment is vibrational relaxation and heat production [53]. Other relevant de-excitation pathway is chemical reaction with suitable cellular substrates [54]. The end result is that 1 O2 is consumed at the same rate that it is produced under biological conditions. So its diffusion distance, directly linked to its lifetime [53], is very short, usually cited much less than a micron. In contrast, H2O2 is a much more stable molecule. Its lifetime is measured in minutes and hours and it has plenty of time to diffuse across the cell [55]. This differences in lifetimes coupled to the different production site can explain the obtained results. With our methodology 1O2 is produced inside the cell (mitochondria and cytoplasm) while H2O2 is being provided from the outside where it diffuses into the different cell compartments. Even after washing, some H2O2 will remain inside the cell and keep diffusing. H2O2 can diffuse from the outside to the cell nucleus by traversing part of the cytoplasm. Then it can give rise to Fenton chemistry in the presence of transition metals (Fe2+/3+, Cu1+/2+) [49]. The outcome of Fenton chemistry is hydroxyl radical generation capable of producing chromatin lesions and, eventually, an increase in micronuclei frequency [41]. On the other hand, given the photosensitized production site of 1O2 and its short lifetime, it seems very improbable that it can reach the nucleus. However, both 1O2 and H2O2 can perturb the cell signalling pathways at the cytoplasm and plasmatic membrane in order to enhance cell proliferation. Although many of the results and proposed mechanisms presented in this work may apply in the field of low level laser (light) therapy (LLLT), we have restricted ourselves purposefully to the classical PDT scheme. In this scheme an exogenous PS (or physiological precursor in our case) is provided to mediate the energy transfer between light and ROS production. Nevertheless, it is clear from our point of view that the same biological principles guide both approaches, low dose PDT and LLLT, in their induction of physiological responses from the cell to the organism levels. This is exemplified in previous works in LLLT such as, for example, that of Lavi et al. where very similar H2O2 concentrations (12–48 lM) to those examined by us were compared to LLLT effect with visible
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light in regard to intracellular Ca2+ levels, cell viability and cell damage in rat cardiomyocytes [56]. 5. Conclusion The results presented indicate that a low dose PDT treatment promotes an increase in cell proliferation and that this cell response is almost equal to that obtained by exposing cell cultures to 10 5 M H2O2 for a short time (10 min). However, the PDT treatment showed no increase in micronuclei frequency while 10 5 M H2O2 gave rise to a statistically significant amount of micronuclei. Accordingly, we propose the use of low dose PDT treatments as a less genotoxic alternative for the study of physiological cell signalling mediated by low ROS concentrations. 6. Abbreviations
GSH H2O2 LLLT MAL MI MTT 1
O2 O2 PDT PE PS ROS
glutathione hydrogen peroxide low level laser (light) therapy methyl 5-aminolevulinate hydrochloride mitotic index 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide singlet oxygen (a1Dg) superoxide anion photodynamic therapy photodynamic effect photosensitizer reactive oxygen species
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