Cytochrome c (Cc) is well-known as an electron carrier at mitochondria, but the hemeprotein can also catalyze peroxidase-like reactions. Although it was previously shown that Cc oxidizes aromatic hydrocarbons and heterocyclic compounds, the reported catalytic efficiencies were low. However, Cc interaction with some lipids increases its peroxidase activity. On this basis, we tested the hypothesis that lipid membranes could improve the study of Cc role in the metabolism of environmental toxicants.

The compounds studied were methyl orange (MO), an azo dye model, and the polycyclic aromatic hydrocarbons (PAHs) benzo[b]fluoranthene and benzo[a]pyrene (BaP). The biotransformation assays were carried out at pH 7.0, with Cc from horse heart at 0.01 mg/mL, in the presence of H₂O₂ at 100 microM. Membranes were prepared as small unilamellar vesicles composed of phosphatidylcholine (PC) or mixtures of PC with cardiolipin (CL) at a 4:1 molar ratio. The transformation of MO was monitored by UV-Vis spectrophotometry and the PAHs measured by HPLC.

The results showed that Cc, in the presence of H₂O₂, slowly transformed MO. The presence of the PC vesicles (200 microM) in the reaction media did not significantly affect the transformation kinetics, but the PC/CL vesicles clearly increased the rate of MO decolorization by Cc. In the case of the PAHs, the Cc-mediated oxidation was almost imperceptible (<15%), in spite of the high compounds’ concentrations used in the assays (1 mg/L). Nevertheless, by including CL vesicles in the assay media, the transformation became evident from the decrease observed in the BaP and BbF chromatographic peaks and by the emergence of reaction products peaks in the chromatograms. Indeed, in the presence of these vesicles, BaP oxidation reached 71±2% after 24 h incubations.

In conclusion, lipid membranes can be employed to further investigate the potential participation of Cc in the metabolism of important toxicants.

Injections of highly cytotoxic or immunomodulating drugs directly into the tumor, is a procedure that is increasingly applied in the clinic. Established Pt-based drugs are used in numerous clinical trials using this administration method. Intratumoral administration may also be advantageous for less stable anticancer metal complexes that fail administration by the standard intravenous route. Particularly, hydrophobic metal-containing complexes are taken up rapidly into cancer cells and cause cell death, while the release of their much less toxic decomposition products into the blood has low overall systemic toxicity and, in some cases may even be beneficial. This concept was originally proposed for hydrophobic vanadium(V) (V(V)) complexes with sterically hindered organic ligands, non-innocent Schiff base V(V) catecholato complexes. Several of these classes of complexes were investigated exhibiting a distinct pattern in their potencies against cancer. These complexes were reactive but sufficiently stable and hence survive under physiological conditions and react with the tumor. This strategy can potentially be applied to other metal complexes, such as titanium(IV), gallium(III) and ruthenium(III) complexes, some of which were previously unsuccessful in human clinical trials when administered via intravenous injections. The intratumoral injection potencies can furthermore be improved by delivery of nanocarrier formulations of potent but unstable metal complexes.

In order to evaluate the immunomodulatory potential of novel monofloral Irish honeys, THP-1 monocyte-derived macrophages provided a suitable cell-based model. THP-1 cells can be differentiated to macrophages using phorbol-12-myristate-13-acetate (PMA). Differentiated cells are then challenged with lipopolysaccharide (LPS) to stimulate the inflammatory cascade. Few studies on the cytotoxic concentrations of these two compounds on THP-1 cells are published. Therefore, the viability of treated THP-1 cells was initially evaluated using trypan blue dye exclusion (PMA), the resazurin assay (LPS), and propidium iodide staining. Furthermore, research to date suggests concentrations ranging from 100 ng/ml down to 15 ng/ml (Lund et al., 2016) and 5 ng/ml (Park et al., 2007) PMA are sufficient for THP-1 differentiation. Consequently, the differentiation potential of this sub-cytotoxic PMA concentration range was also evaluated using flow cytometric detection of the macrophage-specific CD-14 cell surface marker.

The highest concentration of PMA (1 µg/ml) evaluated with the trypan blue exclusion assay caused ~20% cell death. Flow cytometry results indicated CD14 percentage for THP-1 cells exposed to 100ng/ml PMA for 48 hours (~14.63%, SD ± 3.94) were lower than 15ng/ml (~43.41%, SD ± 5.46) and 5ng/ml (~38.36%, SD ±13.12). No substantial time-dependent difference for these concentrations was observed following 48 versus 72-hour exposure. The highest LPS concentration, 100-fold higher than concentrations used in the literature, caused ~29.21% (resazurin assay) and ~25.76% (PI staining) cell death in differentiated THP-1 cells.

In summary, the differentiation capacity of THP-1 cells, and the differentiation ability of the test compounds require cytotoxicity assays to be chosen carefully. Important variables affecting assay outcomes include phenotypic differences between untreated control cells (monocytes) versus treated monocyte-derived macrophages in terms of metabolic and pinocytosis rates.

Dermal exposure to air pollutants is gaining increasing interest at toxicological level. Chlorpyrifos is a broad-spectrum pesticide used for treatments revised by authorities as representing a risk for human health. Few studies investigated the permeation of chlorpyrifos through skin either by using ex vivo animal skin or human skin, but this practice urges for a more ethical mode of action in scientific research.

The purpose of this study was to assess two synthetic membranes as non-animal alternatives to study the dermal permeation of chlorpyrifos for human health risk assessment.

Permeation assays were performed using silicone and Strat-M® membranes mounted on Franz cells with different receptor compositions. Sampling was conducted for HPLC quantification. The permeation kinetic fluxes obtained for the pesticide were calculated and compared to those reported in the literature for in vitro and in vivo human skin.

For both membranes, faster permeation of chlorpyrifos was observed for the highest concentration of ethanol used in the receptor fluid. Therefore, after 8h – work shift time - there was also a higher concentration of the pesticide that permeated both membranes. Regarding the time lag (Tlag), values inferior to 1h were obtained for both membranes and for receptor conditions containing ethanol between 10 and 50%.

Adopting these conditions, the flux values obtained for silicone and Strat-M® membranes were 1.5 ± 0.1 and 1.9 ± 1.2 µg cm^-2h^-1. Importantly, the flux and the Tlag values obtained are close to those reported in human skin studies, supporting the use of these membranes as non-animal systems mimicking the permeation of chlorpyrifos through human skin.

Up to our knowledge, this is the first study where alternative synthetic membranes are used achieving values close to those found for in vivo human exposure to a pesticide, validating experimental conditions to be considered in this research field.

GNE myopathy is an ultra-rare congenital disorder of glycosylation (CDG) that manifests in early adulthood causing progressive distal muscle atrophy and weakness. GNE-CDG results from mutations in the GNE gene, leading to decreased sialic acid (Sia) production [1]. Although hyposialylation has been presumed to be the main cause of GNE-CDG, its pathomechanism may not be exclusively linked to the impaired Sia pathway [2].

Our purpose is to explore cellular and molecular mechanisms that may contribute to GNE-CDG as means of identifying alternative pharmacological targets.

Although immune-mediated responses are not common in GNE-CDG, inflammatory cell infiltration with increased expression of major histocompatibility complex class I (MHC-I) is occasionally reported in muscle biopsies of early-stage GNE-CDG patients [3]. A GNE knockout (KO) cell model was used to evaluate if GNE mutations affect the expression of MHC-I. The results point to higher expression of MHC-I on the surface of GNE-CDG cells. When the GNE KO cells were supplemented with N-acetylmannosamine (ManNAc) and ManNAc-6-phosphate (ManNAc-6-P), intermediates in the Sia biosynthesis, we observed a Sia increase, and a reduction in MHC-I staining. These findings support the hypothesis that Sia content modulates the presence and stability of the MHC-I complex, as previously reported by us [4], and the involvement of a cytotoxic immune response initiated via MHC-I presentation.

Further work is being conducted to better characterize this immunological link, which may contribute to identifying new biomarkers that facilitate GNE-CDG diagnosis and novel therapeutic approaches.

Acknowledgements: UIDP/04378/2020 and UIDB/04378/2020 (UCIBIO), LA/P/0140/2020 (i4HB), and EJP RD COFUND-EJP N 825575 (EJPRD/0001/2020).

References: [1] doi:10.1186/s13023-018-0802-x. [2] doi:10.1016/j.bbrc.2004.12.157. [3] doi:10.1016/S0960-8966(03)00140-8. [4] doi: 10.3390/pharmaceutics12030249.

Polyoxometalates are a well-known group of anionic polynuclear metal oxides presenting important therapeutic potential as anticancer, antiviral, and antibacterial agents [1]. In a previous work [2], we measured the minimum inhibitory concentrations (MIC) of 3 polyoxovanadates (POVs), namely V₁₀, MnV₁₁ and MnV₁₃ against the Gram-negative bacteria Escherichia coli. MICs were obtained following the well-accepted serial two-fold dilution method, which requires a 16-20h culture and might neglect the effects of compounds’ metabolism/speciation changes during the incubation.

In this work, we further studied the antibacterial action of those POVs against the gram-positive Enterococcus faecalis by monitoring the growth of the microorganism at different incubation times.

The compounds were tested at 0.5 mM concentration, based on previous data [2], and growth curves of the bacteria were obtained from the optical density of the cultures at 600 nm. In addition, the MICs of these POVs against E. faecalis were determined for the first time.

The comparison between the growth curves in the presence of the POVs and the corresponding control cultures indicated that the compounds did not affect the lag time of the culture. However, the inhibitory effects of V₁₀, MnV₁₁ and MnV₁₃ on the growth of E. faecalis were evident right from the beginning of the exponential phase. The monomeric species of vanadate (V₁) showed only modest inhibition of bacterial growth. Notably, the inhibition data obtained until 7h culture was sufficient to identify the POVs with stronger antibacterial activity, V₁₀, MnV₁₁ and MnV₁₃, in agreement with the results of MIC determination at 18h. Putting it all together, the results point out that automatized monitoring of the growth curve can meet the need for rapid methods to screen antibacterial POVs against different bacteria.

[1] Aureliano et al. (2021) Coordination Chemistry Reviews, 447, 214143.

[2] Marques-da-Silva et al. (2019) New Journal of Chemistry, 43, 17577.

Chlorpyrifos is a widely used insecticide that has been restricted by regulatory agencies, but still with significant environmental exposure. Nevertheless, few studies investigate the effect of this pesticide in fibroblasts. The aim of this work was to study the effect of chlorpyrifos in the viability of fibroblasts using concentrations detected for human skin exposure. Cellular mechanisms unleashed by chlorpyrifos were also explored.

The GM03349 fibroblast cell line derived from human skin was obtained from the Cell Bank (Coriell Institute) and exposed to different concentrations (0.36-250 µM) of chlorpyrifos and a commercial mixture (Lethal-20), for 6 days. For the viability assays, we used a resazurin assay. For oxidative stress evaluation, after 3h exposure, we measured ROS formation by DCF-DA microplate assay. The inflammatory cytokine IL-6 was measured in the supernatant by ELISA, after 6 days.

For the exposure to chlorpyrifos, only the highest concentration tested (250 µM) resulted in a decrease in cell viability to 18.98%. In the case of Lethal-20, at 125 µM, there was a reduction of cells’ viability to 15.82% and complete cell death at 250 µM. Moreover, ROS production was increased to 1.43 folds and 1.28 folds when the cells were exposed, respectively, to 250 µM chlorpyrifos pure or Lethal-20. To evaluate the inflammatory response, the secretion of IL-6 was assessed in culture supernatants. Exposure to 250 µM chlorpyrifos pure or Lethal-20 gave rise, respectively, to a 1.81 and a 2.43 fold increase of cytokine´s secretion.

In conclusion, prolonged exposure to concentrations of 125 µM in the case of Lethal-20 and to 250 µM in the case of chlorpyrifos caused a significant loss of fibroblasts' viability. Moreover, the toxicity of this pesticide in fibroblasts is evidenced by the induction of oxidative stress and the production of inflammatory cytokines.

The skin is the largest organ of the human body and bioactive compounds like caffeine and catechins have been described for their skin beneficial effects. While caffeine is widely used as a model in skin permeation studies, the skin permeation of catechins is more challenging. Therefore, methods promoting skin absorption of catechins are of great interest.

In this work, we aim to compare the dermal permeation of bioactive compounds through ex vivo pig skin in two situations: 1) in normal condition, without applying any treatment; and 2) in a condition where the skin was pre-treated with a microneedle device.

Permeation assays were performed using ex vivo pig skin mounted on Franz cells. The microneedle device was previously applied to the skin for the treatment condition. Sampling at different time points was conducted for the skin permeation of bioactive compounds. Quantification was calculated using UV spectroscopy and corresponding calibration curves. Additionally, polyphenol content confirmed the results obtained for epicatechin.

Our results showed that higher quantities of the bioactive compounds permeate the skin when the microneedle device was previously applied. For caffeine, after 2h, the pre-treatment promoted a 26 ± 6% increase in the amount permeated compared to untreated skin. A similar increase in the permeation of epicatechin was also observed when the microneedle device was applied. For epicatechin, at 4h, the pre-treatment promoted an increase in the amount permeated superior to 20%, confirmed by UV spectroscopy and by the Folin-Ciocalteu reaction.

Our results show that the microneedle device enhanced the skin permeation of bioactive compounds in terms of quantity permeated, in a similar range for the two compounds tested. Finally, this work encourages further studies of the application of microneedle devices to promote dermal delivery of bioactive compounds.

Abstract: Vanadium is an element with a wide range of effects on the mammalian organism. The ability of this metal to form polyoxovanadates (POVs) and organometallic compounds contributed to the increase in the number of studies on the multidirectional biological activity in view of their application in medicine [1]. Studies of the pharmacological action of vanadium compounds have shown that vanadium has been arousing interest of is potential candidate for therapeutic applications. Many research studies have been shown its anti-bacterial, anti-fungal, anti-parasitic, anti-viral anti-cancer, anti-diabetic, and anti-hypercholesterolemic activity and cardioprotective, neuroprotective, and anti- obesity effects of vanadium compounds. In vitro and in vivo experimental models in several studies have demonstrated the pharmacological potential of vanadium compounds and has shown that some compounds/complexes of this element can be effective against many microbial diseases such as viruses, parasitic protozoan diseases, mycotoxicosis, bacterial diseases caused by gram-negative and gram-positive bacteria such as food poisoning, gastrointestinal, typhoid fever, respiratory infections, tuberculosis, pneumonia, strep throat and skin diseases. It was also shown that certain vanadium compounds/complexes may have potential in anti-cancer, anti-diabetic, and anti-hypercholesterolemic activity and can act as cardioprotective and neuroprotective agents. Thus, from the biological perspective, vanadium compounds have a great potential in the treatment of many types of diseases. However, we must first recognize the therapeutic targets and understand in detail the pharmacokinetics mechanisms (ADME) and pharmacodynamics, for help to design a better and more efficient vanadium-based drugs.

[1] Manuel Aureliano, Nadiia I. Gumerova, Giuseppe Sciortino, Eugenio Garribba, Annette Rompel, Debbie C. Crans, Polyoxovanadates with emerging biomedical activities, Coordination Chemistry Reviews, Volume 447, 2021,214143, ISSN 0010-8545, https://doi.org/10.1016/j.ccr.2021.214143.

Acknowledgments

This study received Portuguese national funds from FCT - Foundation for Science and Technology through projects UIDB/04326/2020, UIDB/04326/2020 and LA/P/0101/2020.

Hydrogel materials have good biomimetic properties and high potential for biomedical and bioanalytical applications. In this work, an hydrogel of serum albumin crosslinked with poly-(ethylene glycol) was prepared and characterized for its water content, protein structure and stability. The ability of the hydrogel to bind small molecule ligands with different hydrophobicity was evaluated using a homologous series of amphiphiles (NBD-Cn, n=4, 6 and 8) and the calculated binding affinities were similar to that of free protein in solution. Overall, the results indicate this type of hydrogel system as convenient tools for studying the binding of xenobiotics to tissue proteins.