Ximena M. Muresan

Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells

Information about the harmful effects of vaping is sparse and inconsistent, therefore, since the use of electronic cigarettes (e-CIGs) has become increasingly popular as a tool to limit tobacco smoking, it is urgent to establish the toxicity of the commercial e-CIGs. Skin (HaCaT) and lung (A549) cells, the main targets of cigarette smoke (CS), were exposed to e-CIG vapor and CS using an in vitro system. The cytotoxic effect of the exposure was analyzed in both cell types by ultrastructural morphology, Trypan Blue exclusion test and LDH assay. In addition, pro-inflammatory cytokines were measured by the Bio-Plex assay. The cytotoxic components of e-CIG were restrained to the flavoring compound and, to a lesser extent, to nicotine although their effects were less harmful to that of CS. Humectants alone exhibited no cytotoxicity but induced the release of cytokines and pro-inflammatory mediators. Based on our results, we can state that exposure to e-CIG vapors results in far less toxic than exposure to CS. In fact, besides the deleterious effect of flavor and nicotine, even the humectants alone are able to evocate cytokines release. This study will hopefully promote the development of safer e-CIGs to help people quit smoking.

Skin Damage Mechanisms Related to Airborne Particulate Matter Exposure.

Epidemiological studies suggest a correlation between increased airborne particulate matter (PM) and adverse health effects. The mechanisms of PM-health effects are believed to involve oxidative stress and inflammation. To evaluate the ability of PM promoting skin tissue damage, one of the main organs exposed to outdoor pollutants, we analyzed the effect of concentrated ambient particles (CAPs) in a reconstructed human epidermis (RHE) model. RHE tissues were exposed to 25 or 100 µg/ml CAPs for 24 or 48 h. Data showed that RHE seems to be more susceptible to CAPs-induced toxicity after 48 h exposure than after 24 h. We found a local reactive O(2) species (ROS) production increase generated from metals present on the particle, which contributes to lipids oxidation. Furthermore, as a consequence of altered redox status, NFkB nucleus translocation was increase upon CAPs exposure, as well as cyclooxygenase 2 and cytochrome P450 levels, which may be involved in the inflammatory response initiated by PM. CAPs also triggered an apoptotic process in skin. Surprisingly, by transition electron microscopy analysis we showed that CAPs were able to penetrate skin tissues. These findings contribute to the understanding of the cutaneous pathophysiological mechanisms initiated by CAPs exposure, where oxidative stress and inflammation may play predominant roles.

Nanostructured lipid carriers (NLC) for the delivery of natural molecules with antimicrobial activity: production, characterisation and in vitro studies

Abstract This study describes the preparation, characterisation and in vitro activity of nanostructured lipid carriers (NLCs) encapsulating natural molecules with antimicrobial activity, such as plumbagin, hydroquinon, eugenol, alpha-asarone and alpha-tocopherol. NLCs were prepared by melt and ultrasonication method, characterised by Cryo-TEM for morphology and SdFFF for dimensional distribution and active encapsulation yields. In vitro tests were conducted on bacteria, fungi and human cell cultures. In vitro tests demonstrated that plumbagin is strongly toxic towards F. oxysporum especially when active molecules are loaded on NLC. Plumbagin was completely non toxic on cyanobacterial model strain up to a threshold over which cell viability was completely lost. NLC loaded with active molecules showed a lower toxicity as compared to their free form on human cultured cells. Although further studies need to be performed, these systems can be potentially proposed to control phytopathogenic organisms.

Modulation of Chloride Currents in Human Lung Epithelial Cells Exposed to Exogenous Oxidative Stress

Air pollution continues to be a major public health concern affecting 9 out of 10 individuals living in urban areas worldwide. Respiratory tract is the organ most exposed to gas pollution, and ozone has been shown to be one of the most noxious pollutants to which living organisms are exposed. In the present work, we have investigated the effects of 0.1 ppm of ozone on chloride currents in human lung epithelial cells (A549 line) and whether this effect could be modulated by vitamin E pre‐treatment. Whole‐cell patch clamp technique was applied to not excitable cells in order to obtain information about chloride currents behavior, important for epithelial lung cells homeostasis. Significant alteration of the I–V curve after ozone treatment was observed, with the appearance of a large outward rectifier component decreasing over time and returning to the basal state levels after 24 h. Statistical analysis indicated a modification of the amount of ions passing the membrane in the unit of time as a possible cause of this difference. RT‐qPCR analysis showed an increase in ClC‐2 and ORCC mRNA after ozone exposure. In addition, pre‐treatment with vitamin E was able to suppress the outward rectifier component induced by ozone, bringing back the current values to the control level and preventing ozone induced chloride channels up regulation. Our data suggest that ozone exposure is able to modify chloride current density and the use of vitamin E can prevent the above‐mentioned damage. J. Cell. Physiol. 232: 1817–1825, 2017.

The Loss of Cellular Junctions in Epithelial Lung Cells Induced by Cigarette Smoke Is Attenuated by Corilagin

Cigarette smoke (CS) contains over 4700 compounds, many of which can affect cellular redox balance through free radicals production or through the modulation of antioxidant enzymes. The respiratory tract is one of the organs directly exposed to CS and it is known that CS can damage the integrity of lung epithelium by affecting cell junctions and increasing epithelium permeability. In this study, we have used a human lung epithelial cell line, Calu-3, to evaluate the effect of CS on lung epithelial cell junctions levels, with special focus on the expression of two proteins involved in intercellular communication: connexins (Cx) 40 and 43. CS exposure increased Cx40 gene expression but not of Cx43. CS also induced NFκB activation and the formation of 4HNE-Cxs adducts. Since corilagin, a natural polyphenol, is able to inhibit NFκB activation, we have determined whether corilagin could counteract the effect of CS on Cxs expression. Corilagin was able to diminish CS induced Cx40 gene expression, 4HNE-Cx40 adducts formation, and NFκB activation. The results of this study demonstrated that CS induced the loss of cellular junctions in lung epithelium, possibly as a consequence of Cx-4HNE adducts formation, and corilagin seems to be able to abolish these CS induced alterations.

Effect of new curcumin-containing nanostructured lipid dispersions on human keratinocytes proliferative responses

This study describes the production and characterization of nanostructured lipid dispersions (NLDs) containing curcumin (CUR) as new tools for curcumin topical delivery. Four types of NLDs based on monoolein in association with different emulsifiers were produced: Na cholate and poloxamer 407 (NLD1), poloxamer alone (NLD2), the mixture of Na cholate and Na caseinate (NLD3) and Na cholate alone (NLD4). Morphology and dimensional distribution of lipid dispersions were investigated by cryo‐TEM and photon correlation spectroscopy (PCS). In vitro studies based on Franz cell, membrane nylon and stratum corneum–epidermis (SCE) were carried out to compare the four NLDs in terms of cytotoxicity in human keratinocytes and CUR diffusion. Our PCS studies showed differences in particles diameter among the different NLDs. In addition, cytotoxicity results in HaCaT cells evidenced that NLD1 and NLD2 were toxic at doses over 1 μm. Therefore, cryo‐TEM was determined only for NLD3 and NLD4 showing that CUR did not affect their structure. Diffusion measurement in SCE and nylon membrane evidenced that CUR had a time‐delayed release for NLD4. The ‘wound healing’ effect of NLD3 and NLD4 with and without CUR analysed keratinocytes in vitro, and a clear inhibition of cell proliferation/migration by CUR was observed. This effect was mediated by the inhibition of cyclin D1 expression as a consequence of the impaired NFkB activation. This study confirms the antiproliferative properties of CUR and evidenced a new possible model of CUR topical delivery for hyperproliferative cutaneous diseases such as psoriasis.

SRB1 as a new redox target of cigarette smoke in human sebocytes

Abstract For its critical location, the skin represents the major interface between the body and the environment, therefore is one of the major biological barriers against the outdoor environmental stressors. Among the several oxidative environmental stressors, cigarette smoke (CS) has been associated with the development and worsening of many skin pathologies such as acne, dermatitis, delayed wound healing, aging and skin cancer. In our previous work we have demonstrated that CS is able to affect genes involved in skin cholesterol trafficking, among which SRB1, a receptor involved in the uptake of cholesterol from HDL, seems to be very susceptible to the oxidative stress induced by CS. In the present work we wanted to investigate the presence of SRB1 in human sebocytes and whether CS can affect cholesterol cellular uptake via the redox modulation of SRB1. By using a co‐culture system of keratinocytes/sebocytes, we found that CS exposure induced a SRB1 protein loss without affecting sebocytes viability. The decrease of SRB1 levels was a consequence of SRB1/HNE adducts formation that leads to SRB1 ubiquitination and degradation. Moreover, the CS‐induced loss of SRB1 induced an alteration of sebocytes lipid content, also demonstrated by cholesterol quantification in SRB1 siRNA experiments. In conclusion, exposure to CS, induced SRB1 post‐translational modifications in sebocytes and this might affect sebocytes/skin functionality. Graphical abstract Figure. No Caption available. HighlightsSebocytes are involved in maintaining skin functions.Skin is the first barrier against outdoor insults.Cigarette smoke (CS) affects skin homeostasis.CS decreases SRB1 levels compromising sebocytes cholesterol uptake.Lipids are essential to keep skin functionality.

Keratinocytes oxidative damage mechanisms related to airbone particle matter exposure

Epidemiological evidences have correlated airbone particulate matter (PM) to adverse health effects, mainly linking to pulmonary and cardiovascular disease. Nevertheless, only recently, some studies reported detrimental effects of PM on other organs such as skin. In a recent work, we have reported increased oxidative and inflammatory responses in Reconstituted Human Epidermis (RHE) exposed to ambient particles (CAPs) and we also demonstrated the ability of CAPs to penetrate the skin tissue. The present study was aimed to better understand the cellular mechanisms beyond the oxidative changes induced by CAPs (5-10-25μg/mL) in human immortalized keratinocytes (HaCaT). After 24h of treatment, CAPs were able to enter the cells leading to a decrease in viability, increased levels of 4-hydroxinonenal products (4-HNE) and IL-1α release. Overall these data, suggest lipid and protein oxidative damage, as well as an increase of inflammatory response after being challenged with CAPs. In addition, 3h after CAPs exposure we found a significant increase in NF-kB and Nrf2 translocation into the nucleus. In contrast, no differences in gene expression and enzymatic activity of Nrf2 target genes were detected. This last finding could be explained by the ability of CAPs to possibly alter the binding of Nrf2 to the ARE DNA sequence.

Antitubulinic effect of New Fluorazone Derivatives on Melanoma Cells

Microtubules are composed by α- and β-tubulin polypeptides. α-tubulin undergoes a reversible posttranslational modification whereby the C-terminal tyrosine residue is removed (Glu-tubulin) and re-added (Tyrtubulin). Recent studies have shown that α-tubulin tyrosine residues can be nitrated and the incorporation of NO2Tyr into the C-terminus of Glu-tubulin forms a complex that blocks the tyrosination/detyrosination cycle, an event that can compromise protein/enzyme functions, such as cell division. Since many studies demonstrated that Glu-tubulin levels increase in cancer, the aim of the present study was to investigate the effect of new drugs, fluorazone derivatives (K1-K2-K9-K10-K11), on the proliferation of melanoma cells. Our results demonstrated that these drugs, except for K2, were able to inhibit cellular proliferation without exhibiting cytotoxicity. The anti-proliferative effect was accompanied by the decrease of Glu-tubulin levels and the increase of its nitration. This effect seems to be a consequence of NO2 induction and NO2Tyr ligation to Glu-tubulin. Collectively, these results, showing that the fluorazone derivatives, by promoting NO2Tyr incorporation into α-tubulin, are able to arrest the cycle of detyrosination/tyrosination and to inhibit cell proliferation, offer new perspectives for the possible usage of these drugs, alone or in combination, as non-toxic, anti-proliferative agents in melanoma.

SR-B1 involvement in keratinocytes in vitro wound closure

Skin represents the most extended organ of human body, having as main function the protection of our body from outdoor stressors. Its protective ability is compromised when the skin is disrupted as a consequence of mechanical insults. For this purpose, cutaneous tissue is equipped with an efficient and fine mechanism involved in repairing the wounded area. Among the numerous players that take part in the wound healing process, SR-B1 has been recently shown to have a role in keratinocyte re-epithelialization. SR-B1 is a mediator of cholesterol uptake from HDLs, whereas it is implicated in other cellular processes such as vitamins absorption, vesicle trafficking or pathogen identification. The aim of this study was to investigate the mechanisms involved in SR-B1 role in skin wound closure. Our in vitro data demonstrated that SR-B1 influenced keratinocyte proliferation and migration through a downregulation of nuclear cyclin D1 levels and active MMP9 expression respectively possibly in an NF-kB-dependent mechanism. In addition, SR-B1 was also able to modulate keratinocyte morphology into a pro-migratory cytoskeleton rearrangement. The present in vitro study suggests a new role of SRB1 as a possible new key player in cutaneous wound healing mechanism.