Chemical features of the photosensitizers new methylene blue N and S137 influence their subcellular localization and photoinactivation efficiency in Candida albicans.

Antimicrobial photodynamic treatment (APDT) has emerged as an effective therapy against pathogenic fungi with both acquired and intrinsic resistance to commonly used antifungal agents. Success of APDT depends on the availability of effective photosensitizers capable of acting on different fungal structures and species. Among the phenothiazinium dyes tested as photoantifungals, new methylene blue N (NMBN) and the novel pentacyclic compound S137 are the most efficient. In the present study we compared the effects of APDT with NMBN and S137 on the survival of Candida albicans and employed a set of fluorescent probes (propidium iodide, FUN-1, JC-1, DHR-123 and DHE) together with confocal microscopy and flow cytometry to evaluate the effects of these two chemically diverse photosensitizers on cell membrane permeability, metabolism and redox status, and mitochondrial activity. Taken together, our results indicate that, due to chemical features resulting in different lipophilicity, NMBN and S137 localize to distinct subcellular structures and hence inactivate C. albicans cells via different mechanisms. S137 localizes mostly to the cell membrane and, upon light exposure, photo-oxidizes membrane lipids. NMBN readily localizes to mitochondria and exerts its photodynamic effects there, which was observed to be a less effective way to achieve cell death at lower light fluences.

Photodynamic inactivation of Candida albicans and Candida tropicalis with aluminum phthalocyanine chloride nanoemulsion.

The in vitro susceptibilities of Candida albicans and Candida tropicalis to Antimicrobial Photodynamic Treatment with aluminum phthalocyanine chloride in nanoemulsion (ClAlPc/NE) were investigated. PS concentration- and fluence-dependent cell survival after APDT were compared before and after unbound extracellular PS had been washed out. The PS uptake and its subcellular localization were also determined. Exposure to light in the absence of the PS and treatment with the PS in the absence of light did not kill the fungi. APDT with ClAlPc/NE resulted in a reduction of five orders of magnitude in viability for C. albicans and between four and five orders of magnitude for C. tropicalis. Washing the cells to remove unbound PS before light exposure did not impair fungal inactivation, suggesting that cell photosensitization was mainly carried out by cell bound ClAlPc. The degree of ClAlPc uptake was dependent on its concentration. Internalization of ClAlPc by C. albicans and C. tropicalis was confirmed by confocal fluorescence microscopy that showed the PS does not penetrate the nucleus and instead accumulates in specific regions of the cytoplasm. Our results show that incorporating the water-insoluble ClAlPc into a nanoemulsion leads to an efficient formulation capable of photoinactivating both Candida species.

The effects of photodynamic treatment with new methylene blue N on the Candida albicans proteome.

Candida albicans is a human pathogenic fungus mainly affecting immunocompromised patients. Resistance to the commonly used fungicides can lead to poor treatment of mucosal infections which, in turn, can result in life-threatening systemic candidiasis. In this scenario, antimicrobial photodynamic treatment (PDT) has emerged as an effective alternative to treat superficial and localized fungal infections. Microbial death in PDT is a consequence of the oxidation of many cellular biomolecules, including proteins. Here, we report a combination of two-dimensional electrophoresis and tandem mass spectrometry to study the protein damage resulting from treating C. albicans with PDT with new methylene blue N and red light. Two-dimensional gels of treated cells showed an increase in acidic spots in a fluence-dependent manner. Amino acid analysis revealed a decrease in the histidine content after PDT, which is one plausible explanation for the observed acidic shift. However, some protein spots remained unchanged. Protein identification by mass spectrometry revealed that both modified and unmodified proteins could be localized to the cytoplasm, ruling out subcellular location as the only explanation for damage selectivity. Therefore, we hypothesize that protein modification by PDT is a consequence of both photosensitizer binding affinity and the degree of exposure of the photooxidizable residues on the protein surface.

Potential risks of the residue from Samarco's mine dam burst (Bento Rodrigues, Brazil).

On November 5th, 2015, Samarco's iron mine dam - called Fundão - spilled 50-60 million m3 of mud into Gualaxo do Norte, a river that belongs to Rio Doce Basin. Approximately 15 km2 were flooded along the rivers Gualaxo do Norte, Carmo and Doce, reaching the Atlantic Ocean on November 22nd, 2015. Six days after, our group collected mud, soil and water samples in Bento Rodrigues (Minas Gerais, Brazil), which was the first impacted area. Overall, the results, water samples - potable and surface water from river - presented chemical elements concentration according to Brazilian environmental legislations, except silver concentration in surface water that ranged from 1.5 to 1087 µg L-1. In addition, water mud-containing presented Fe and Mn concentrations approximately 4-fold higher than the maximum limit for water bodies quality assessment, according to Brazilian laws. Mud particle size ranged from 1 to 200 µm. SEM-EDS spot provided us some semi quantitative data. Leaching/extraction tests suggested that Ba, Pb, As, Sr, Fe, Mn and Al have high potential mobilization from mud to water. Low microbial diversity in mud samples compared to background soil samples. Toxicological bioassays (HepG2 and Allium cepa) indicated potential risks of cytotoxicity and DNA damage in mud and soil samples used in both assays. The present study provides preliminary information aiming to collaborate to the development of future works for monitoring and risk assessment.