Cytotoxic effect and apoptosis pathways activated by methylene blue-mediated photodynamic therapy in fibroblasts

https://doi.org/10.1016/j.pdpdt.2020.101654Get rights and content

Highlights

  • MB-mediated aPDT induced a significant dose-dependent cytotoxic effect on mouse fibroblasts.

  • The apoptosis mechanisms were related to mitochondrial photodamage.

  • The cytotoxicity occurred by the activation of Bcl-2 apoptotic genes.

Abstract

Antimicrobial photodynamic therapy (aPDT) has been used as an adjuvant treatment of oral infections as a minimal intervention clinical approach. Its antimicrobial efficacy was demonstrated in several studies; however, there is a lack of evidence on its cytotoxic effect on mouse fibroblasts (NIH/3T3). The aim of this study was to evaluate the cytotoxicity and apoptotic pathways of methylene blue-mediated aPDT on mouse fibroblasts. Cells were treated with 0.1 or 1.0 mg.L−1 methylene blue (MB), and 0.075 or 7.5 J.cm-² LED at 630 nm. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while cDNA expression for Bax, Bad, Bcl-2, VDAC-1, cytochrome C and Fas-L was assessed by qRT-PCR (1, 3, 6 and 24 h). The differences between groups were detected by Kruskal-Wallis and post-hoc Dunn's tests for MTT and CV assays, and by ANOVA and post-hoc Tukey test for qPCR (P < 0.05). The combination of 1.0 mg.L−1 MB and 7.5 J.cm-² LED significantly reduced the cellular viability, whereas MB and LED alone were innocuous to fibroblasts. MB-mediated aPDT increased the expression of cytochrome C and Fas-L after 3 h, and Bax/Bcl-2, Bad/Bcl-2, and VDAC-1 after 6 h from treatment. Based on these results, MB-mediated aPDT induced cytotoxicity on mouse fibroblasts, with consequent activation of Bcl-2 apoptosis signaling pathways. Further studies are needed to determine the adequate parameters of aPDT to inactivate microorganisms without damaging fibroblasts.

Introduction

Minimal intervention approaches have been advocated in dental procedures for treating oral diseases, reducing the risks of irreversible damages of tooth structure with maximum comfort and cost-effectiveness for patients. In this sense, antimicrobials are being applied into cavitated caries lesions to reduce the number of viable pathogenic microorganisms, without the need of removing mineralized tissues [[1], [2], [3], [4], [5]]. An option commonly employed in this field is antimicrobial photodynamic therapy (aPDT), which promotes the inactivation and/or death of microorganisms by the association of three components, a chemical photosensitizing agent (PS), a complementary light source and molecular oxygen [[4], [5], [6]].

Methylene blue (MB) [chloride 3.7 bis(dimethylamino) phenothiazine-5-io] is a dye from the phenothiazinium class of compounds [9], with favorable properties for aPDT, such as low molecular weight, classical 1O2 generator, hydrophilicity, cationic form at physiological pH and strong light absorption at 660 nm [[7], [8], [9], [10]]. Briefly, molecules of MB incorporated to cellular structures of microorganisms are able to absorb photons and excite electrons. These electrons are transferred to a substrate or molecular oxygen, resulting in superoxides that can damage or kill microbial cells [5,11].

We have previously demonstrated the efficacy of 100 mg.L−1 MB associated with 75 J.cm-2 LED on dentin caries microcosms [12]. However, the evidence of aPDT on eukaryotic cytotoxic effects of this therapeutic method is still scarce. A previous study showed that 10 μmol.L−1 MB activated with 36 J.cm-2 light energy did not produce a significant cytotoxicity on mouse fibroblasts (L929) [13]. Similarly, MB-mediated aPDT in different concentrations (2.5, 12.5 and 25 μg/ml) did not increase cytotoxicity in human fibroblasts compared to no treatment groups [14,15]. Also, 1 μM of MB combined with 53.4 J.cm-2 light energy did not affect the mitochondrial activity of human gingival fibroblasts and human osteoblasts in vitro [16].

Considering that previous studies [[13], [14], [15], [16]] have tested a very low concentration of MB, which may not be efficient against biofilms, the aim of this in vitro study was to evaluate the cytotoxicity and apoptotic effects of methylene blue-mediated antimicrobial photodynamic therapy, using different MB concentrations and light energies [12] on mouse fibroblasts (NIH/3T3). The null hypotheses were that aPDT would have no cytotoxicity (H0) and apoptotic effects (H0’) on eukaryotic cells, regardless of the dose.

Section snippets

Cell culture

NIH/3T3 mouse fibroblasts cells (ATCC - American Type Culture Collection, CRL1658™) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, USA) supplemented with 10 % (v/v) fetal bovine serum (FBS, GIBCO Laboratories, Life Technologies, Inc., New York, USA), 100 IU. mL−1 penicillin and 0.1 mg.mL−1 streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % air. The cells were enzymatically dissociated with trypsin (0.25 % trypsin, 1 mmol.L−1

Results

MB-mediated aPDT demonstrated a significant cytotoxicity effect in MTT assay. Cytotoxic effects were not detected by the application of LED alone and lower concentrations of MB-mediated aPDT, in comparison to control group. The association of 0.1 mg.L−1 MB and 7.5 J.cm−2 LED reduced the viability of fibroblasts at 24 h, while 1 mg.L−1 MB and 7.5 J.cm-2 produced a significant reduction of viability in all times (33.3–63.2 %). The highest reduction of cellular viability was observed after

Discussion

These findings indicate that MB-mediated aPDT promoted cytotoxicity in mouse fibroblasts, reducing adherent cells with the increment of MB and light doses. This antimicrobial therapy stimulated cellular apoptosis, increasing the relative expression of Bax/Bcl-2, Bad/Bcl-2, VDAC-1 and cytochrome C. Although MB and LED alone did not produce considerable cellular cytotoxicity, aPDT with its highest parameters of concentration and energy density decreased the fibroblasts viability significantly;

Acknowledgements

The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) for funding this research (grant# 132411/2017-2), and Dr. Carlos Ferreira Santos for providing the infrastructure and reagents for qRT-PCR assay.

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