Photosensitizers attenuate LPS-induced inflammation: implications in dentistry and general health.

Antimicrobial photodynamic therapy (aPDT) is a complementary therapeutic modality for periodontal and endodontic diseases, in which Gram-negative bacteria are directly involved. Currently, there are few evidences regarding the effects of aPDT on bacterial components such as lipopolysaccharide (LPS) and it would represent a major step forward in the clinical use of this therapy. In this context, this study aimed to evaluate the efficacy of different photosensitizers (PSs) used in aPDT in LPS inhibition. Four PSs were used in this study: methylene blue (MB), toluidine blue (TBO), new methylene blue (NMB), and curcumin (CUR). Different approaches to evaluate LPS interaction with PSs were used, such as spectrophotometry, Limulus amebocyte lysate (LAL) test, functional assays using mouse macrophages, and an in vivo model of LPS injection. Spectrophotometry showed that LPS decreased the absorbance of all PSs used, indicating interactions between the two species. LAL assay revealed significant differences in LPS concentrations upon pre-incubation with the different PSs. Interestingly, the inflammatory potential of LPS decreased after previous treatment with the four PSs, resulting in decreased secretion of inflammatory cytokines by macrophages. In vivo, pre-incubating curcumin with LPS prevented animals from undergoing septic shock within the established time. Using relevant models to study the inflammatory activity of LPS, we found that all PSs used in this work decreased LPS-induced inflammation, with a more striking effect observed for NMB and curcumin. These data advance the understanding of the mechanisms of LPS inhibition by PSs.

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.

Biological and In silico Studies on Synthetic Analogues of Tyrosine Betaine as Inhibitors of Neprilysin - A Drug Target for the Treatment of Heart Failure.

BACKGROUND: Fungal secondary metabolites are important sources for the discovery of new pharmaceuticals, as exemplified by penicillin, lovastatin and cyclosporine. Searching for secondary metabolites of the fungi Metarhizium spp., we previously identified tyrosine betaine as a major constituent. METHODS: Because of the structural similarity with other inhibitors of neprilysin (NEP), an enzyme explored for the treatment of heart failure, we devised the synthesis of tyrosine betaine and three analogues to be subjected to in vitro NEP inhibition assays and to molecular modeling studies. RESULTS: In spite of the similar binding modes with other NEP inhibitors, these compounds only displayed moderate inhibitory activities (IC50 ranging from 170.0 to 52.9 µM). However, they enclose structural features required to hinder passive blood brain barrier permeation (BBB). CONCLUSIONS: Tyrosine betaine remains as a starting point for the development of NEP inhibitors because of the low probability of BBB permeation and, consequently, of NEP inhibition at the Central Nervous System, which is associated to an increment in the Aß levels and, accordingly, with a higher risk for the onset of Alzheimer's disease.

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.

Genetic polymorphisms in glutathione (GSH-) related genes affect the plasmatic Hg/whole blood Hg partitioning and the distribution between inorganic and methylmercury levels in plasma collected from a fish-eating population.

This study aims to evaluate the effects of polymorphisms in glutathione (GSH-) related genes (GSTM1, GSTT1, GSTP1, GCLM, and GCLC) in the distribution of Hg in the blood compartments in humans exposed to methylmercury (MeHg). Subjects (n = 88), exposed to MeHg from fish consumption, were enrolled in the study. Hg species in the plasma compartment were determined by LC-ICP-MS, whereas genotyping was performed by PCR assays. Mean total Hg levels in plasma (THgP) and whole blood (THgB) were 10 ± 4.2 and 37 ± 21, whereas mean levels of plasmatic MeHg (MeHgP), inorganic Hg (IHgP), and HgP/HgB were 4.3 ± 2.9, 5.8 ± 2.3 µg/L, and 0.33 ± 0.15, respectively. GSTM1 and GCLC polymorphisms influence THgP and MeHgP (multivariate analyses, P < 0.050). Null homozygotes for GSTM1 showed higher THgP and MeHgP levels compared to subjects with GSTM1 (THgP ß = 0.22, P = 0.035; MeHgP ß = 0.30, P = 0.050) and persons carrying at least one T allele for GCLC had significant higher MeHgP (ß = 0.59, P = 0.046). Also, polymorphic GCLM subjects had lower THgP/THgB than those with the nonvariant genotype. Taken together, data of this study suggest that GSH-related polymorphisms may change the metabolism of MeHg by modifying the distribution of mercury species iin plasma compartment and the HgP/HgB partitioning.

Polymorphisms in glutathione-related genes modify mercury concentrations and antioxidant status in subjects environmentally exposed to methylmercury.

Methylmercury (MeHg) toxicity may vary widely despite similar levels of exposure. This is hypothetically related to genetic differences in enzymes metabolizing MeHg. MeHg causes oxidative stress in experimental models but little is known about its effects on humans. The aims of the present study was to evaluate the effects of polymorphisms in glutathione (GSH)-related genes (GSTM1, GSTT1, GSTP1 and GCLM) on Hg concentrations in blood and hair, as well as MeHg-related effects on catalase (CAT) and glutathione-peroxidase (GPx) activity and GSH concentrations. Study subjects were from an Amazonian population in Brazil chronically exposed to MeHg from fish. Hg in blood and hair were determined by ICP-MS, CAT, GPx and GSH were determined by spectrophotometry, and multiplex PCR (GSTM1 and GSTT1) and TaqMan assays (GSTP1 and GCLM) were used for genotyping. Mean Hg concentrations in blood and hair were 48±36 µg/L and 14±10 µg/g. Persons with the GCLM-588 TT genotype had lower blood and hair Hg than did C-allele carriers (linear regression for Hg in blood ß=-0.32, p=0.017; and hair ß=-0.33; p=0.0090; adjusted for fish intake, age and gender). GSTM1*0 homozygous had higher blood (ß=0.20; p=0.017) and hair Hg (hair ß=0.20; p=0.013). Exposure to MeHg altered antioxidant status (CAT: ß=-0.086; GSH: ß=-0.12; GPx: ß=-0.16; all p<0.010; adjusted for gender, age and smoking). Persons with GSTM1*0 had higher CAT activity in the blood than those with GSTM1. Our data thus indicate that some GSH-related polymorphisms, such as GSTM1 and GCLM may modify MeHg metabolism and Hg-related antioxidant effects.