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.