Intense Pulsed Light: Friend or Foe? Molecular Evidence to Clarify Doubts

Materials and Methods

Animal protocol. Twenty-eight female mice (Mus musculus, ICR (CD-1®)) with 5 weeks old were obtained from Harlan Interfauna (Barcelona, Spain). After 1 week of acclimatization, the animals were randomly divided into five experimental groups: Control (n=5); DMBA (n=5); DMBA+TPA (n=6); DMBA+IPL (n=6); IPL (n=6). Animals were housed in plastic cages, with hard wood chips for bedding, and maintained on a basal diet (4RF21®, Mucedola, Settimo Milanese, Milan, Italy). The room temperature and the relative humidity were controlled at 23±2°C and 50±10%, respectively. The following protocol was approved by the Portuguese Ethics Committee for Animal Experimentation and was performed in accordance to the National and European Community Legislation for Animal Welfare (National Decree-Law 113/2013 and European Directive 2010/63/EU).

Animal protocol was initiated with the hair cutting of the mice dorsum region to be exposed to chemical agents and/or IPL. The hair cut was made with a shearing machine (Aesculap GT420 Isis, Aesculap Inc., Center Valley, PA, USA). In the animals from groups DMBA, DMBA+IPL and DMBA+TPA, it was topically applied a single dose of the initiator agent 12-dimethylbenz(a)anthracene (DMBA, 2 mM prepared in acetone). Four days after the application of DMBA, 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 mM prepared in acetone) was applied to the animals from DMBA+TPA group twice a week for 25 weeks. The animals from groups DMBA+IPL and IPL were exposed to intense pulsed light (Lumea SC2001/00, Phillips, Amsterdam, Netherlands), 2 pulses at an intensity of 5, which correspond to 6.5 J/cm² in an area of 6 cm², twice a week and throughout the animal protocol. IPL pulses were applied parallel to the sagittal line of the mice. Acetone was topically applied, twice a week, to the skin of the animals from control group. Animals were observed daily for health check.

At the end of the experimental protocol, animals were euthanized with ketamine/xylazine (Imalgene® 1000, Merial S.A.S., Lyon, France and Rompun® 2%, Bayer Healthcare S.A., Kiel, Germany, respectively) and blood was collected. All noticeable tumors in the skin region submitted to the tested treatments were counted and removed for biochemical and histologic analysis.

Skin histologic analysis. Cubic pieces from skin tissue were fixed [4% (v/v) buffered paraformaldehyde] by diffusion during 24 h and subsequently dehydrated with graded ethanol and included in paraffin blocks. Serial sections (2 μm of thickness) of paraffin blocks were cut by a microtome (Leica®20035, Leica Microsystems, Wetzlar, Germany). Deparaffinized sections of skin tissue were stained for hematoxylin-eosin and classified according to Thomas-Ahner 2007 (7).

Blood tests. Serum albumin, total protein, the activity the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in duplicate on an AutoAnalyzer (Prestige 24i, Cormay PZ, Diamond Diagnostics, Holliston, MA, USA). Serum C-reactive protein and IL-6 levels were assayed by immunoblotting as described below.

Preparation of skin samples for biochemical analysis. Skin specimens homogenized in solubilization buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.1% PMSF) in the ratio of 50 mg tissue per ml of solubilization buffer. The homogenization was performed with a Potter-Elvehjem homogenizer and a Teflon pestle. All the procedures were performed on ice or below 4°C. Protein content was determinate with the RD DC Protein Assay Kit (Bio-Rad, Holliston, CA, USA).

Immunoblotting analysis. For the analysis of CRP and IL-6, serum samples were diluted 1:20 in TBS and a volume of 100 μl was slot-blotted into a nitrocellulose membrane. For the analysis of metabolic proteins, equivalent amounts of skin tissue proteins of each group were electrophoresed on a 12.5% SDS-PAGE. Gels were blotted onto a nitrocellulose membrane (Whatman®, Protan®) in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) during 2 h (200 mA). For the analysis of 3-nitrotyrosine, skin extracts were diluted in TBS to obtain a final protein concentration of 0.001 μg/μl and a volume of 100 μl was slot-blotted into a nitrocellulose membrane. Then, nonspecific binding was blocked with 5% (w/v) dry nonfat milk in TBS-T (100 mM Tris, 1.5 mM NaCl, pH 8.0 and 0.5% Tween 20). Membranes with serum samples were incubated with primary antibody diluted 1:1000 5% (w/v) nonfat dry milk in TBS-T (anti-C-reactive protein (CRP; ab32412, Abcam, Cambridge, UK) or anti-Interleukin-6 (IL-6; ab6672, Abcam, Cambridge, UK)) for 2 h at room temperature, washed and incubated with secondary horseradish peroxidase-conjugated anti-rabbit (1:1,000; GE Healthcare, Milwaukee, WI, USA). Membranes with skin extracts were incubated with primary antibody diluted 1:1000 in 5% (w/v) non-fat dry milk in TBS-T (anti-GAPDH (ab9485, Abcam, Cambridge, UK), anti-ATP synthase subunit β (ab14730, Abcam, Cambridge, UK), anti-p53 (Sc99, Santa Cruz, Dallas, Tx, USA), anti-cleaved caspase-3 (PC679, CalbioChem, San Diego, CA, USA), anti-3-nitrotyrosine (06-284, Merck Millipore, Darmstadt, Germany)) for 2 h at room temperature, washed and incubated with secondary horseradish peroxidase-conjugated anti-rabbit (GE Healthcare, Milwaukee, WI, USA), respectively. Immunoreactive bands were detected by enhanced chemiluminescence ECL (Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to the manufacturer's procedure and images were recorded using X-ray films (Kodak Biomax Light Film, Sigma, MO, USA). The films were scanned in Molecular Imager Gel Doc XR+ System and analyzed with QuantityOne software (v4.1, Bio-Rad, Holliston, CA, USA). Control for protein loading was confirmed by Ponceau S staining.

For the protein carbonyl derivate assay, a given volume (V) of sample containing 20 μg of protein was derivatized with 2,4-dinitrophenylhydrazine (DNPH). Briefly, the sample was mixed with 1V of 12% sodium dodecyl sulfate, 2V of 2 mM DNPH/10% trifluoroacetic acid, followed by 30 minutes of incubation in the dark, after which 1.5 V of 2 M Tris-base/18.3% of β-mercaptoethanol was added for neutralization. After diluting the derivatized proteins in TBS to obtain a final concentration of 0.001 μg/μl, a 100 μl volume was slot-blotted into a nitrocellulose membrane. Immunodetection was performed as above-referred with anti-DNP (1:1000, V041, DakoCytomation, Glostrup, Denmark) as primary antibody.

2DE-blot-MS/MS analysis. In brief, 150 μg proteins of skin extracts were diluted to 125 μl with a rehydration solution containing 8 M urea, 2 M thiourea, 1% CHAPS, 12.9 mM DTT, 1% ampholytes 3-10; 0.01% of blue bromophenol and loaded on 7 cm IPG strips (pH 3-10 NL; GE Healthcare, Milwaukee, WI, USA) with a pH 3-10. The isoelectric separation was performed using the following focusing program: 12 h at 50 mV in rehydration, 2 h at 150 V (gradient), 1 h at 500 V (gradient), 1 h at 1,000 V (gradient) and 1.5 h at 5,000V (“step-n-hold”). After IEF separation, the gel strips were applied on top of a 12.5% SDS-PAGE and proteins were separated at 150 V constant current until the bromophenol blue front reached the bottom of the gel. Gels were stained with BlueSafe (Nztech) or were blotted onto a nitrocellulose membrane (Whatman®, Protan®) in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) during 2 h (200 mA).

For the analysis of carbonylated proteins, before SDS-PAGE, strips were incubated with 5 ml of 12% SDS, during 20 min, followed by incubation with 5 ml of 20 mM DNPH solution, during 1 h in the dark with agitation. Then, strips were incubated for 30 min with a solution buffer containing 2% (w/v) SDS, 6 M urea, 30% glycerol, 0.05 M Tris-HCl pH 8,8 and 20 mg/ml DTT.

Immunodetection of carbonylated and nitrated proteins was performed as above described. Gels and films were scanned into the Gel Doc XR + System (Bio-Rad, Holliston, CA, USA) and analyzed using PDQuest software (v7.2, Bio-Rad, Holliston, CA, USA).

The protein spots immunostained with anti-DNP or with anti-3-nitrotyrosine were removed from the 2D-gel, destained with 25 mM ammonium bicarbonate/50% acetonitrile, and dried in vacuum concentrator (SpeedVac, Thermo Savant). The dried gel pieces were rehydrated with 25 μl of 10 μg/ml trypsin in 50 mM ammonium bicarbonate and digested overnight at 37°C. Tryptic peptides were dried in a vacuum concentrator and resuspended in 10 μl of a 50% acetonitrile/0.1% formic acid solution. MS was performed on a matrix-assisted laser desorption/ionization-time-of-flight MALDI-TOF/TOF mass spectrometer (4800 Proteomics Analyzer, AbSCIEX) in the positive ion reflector mode. The ten most intense peaks in each spot were selected for further perform of MS/MS spectra, excluding the peaks of trypsin autolysis or of acrylamide. The spectra were processed and analyzed by Global Protein Server Workstation (Applied Biosystems, Foster City, CA, USA), which uses as Mascot search engine (Matrix Science, London, UK), for the identification of proteins, combining data from PMF (peptide mass fingerprinting) with MS/MS data for research in an internal database. The protein identification was considered when the degree of confidence was higher than 98%.

Statistical analysis. The variables analyzed showed a normal distribution, according to the Kolmogorov-Smirnov test. For this reason, the results obtained from biochemical analyses were processed using parametric statistical tests. Differences between experimental groups were analyzed by one-way ANOVA followed by Tukey post-hoc test for multiple comparisons. The calculations were performed with the software GraphPad Prism (version 5.0, La Jolla, CA, USA). The values obtained were considered statistically significant when p<0.05. The results for all variables are presented as mean±standard deviation.