Assessment of the impact of direct in vitro PFAS treatment on mouse spermatozoa.

Abstract: Poly- and per-fluoroalkyl substances (PFAS) are synthetic environmentally persistent chemicals. Despite the phaseout of specific PFAS, their inherent stability has resulted in ubiquitous and enduring environmental contamination. PFAS bioaccumulation has been reported globally with omnipresence in most populations wherein they have been associated with a range of negative health effects, including strong associations with increased instances of testicular cancer and reductions in overall semen quality. To elucidate the biological basis of such effects, we employed an acute in vitro exposure model in which the spermatozoa of adult male mice were exposed to a cocktail of PFAS chemicals at environmentally relevant concentrations. We hypothesized that direct PFAS treatment of spermatozoa would induce reactive oxygen species generation and compromise the functional profile and DNA integrity of exposed cells. Despite this, post-exposure functional testing revealed that short-term PFAS exposure (3 h) did not elicit a cytotoxic effect, nor did it overtly influence the functional profile, capacitation rate, or the in vitro fertilization ability of spermatozoa. PFAS treatment of spermatozoa did, however, result in a significant delay in the developmental progression of the day 4 pre-implantation embryos produced in vitro. This developmental delay could not be attributed to a loss of sperm DNA integrity, DNA damage, or elevated levels of intracellular reactive oxygen species. When considered together, the results presented here raise the intriguing prospect that spermatozoa exposed to a short-term PFAS exposure period potentially harbor an alternate stress signal that is delivered to the embryo upon fertilization. Lay summary: PFAS are synthetic chemicals widely used in non-stick cookware, food packaging, and firefighting foam. Such extensive use has led to concerning levels of environmental contamination and reports of associations with a spectrum of negative health outcomes, including testicular cancer and reduced semen quality. To investigate the effects of PFAS on male reproduction, we incubated mouse sperm in a cocktail of nine PFAS at environmentally relevant concentrations before checking for a range of functional outcomes. This treatment strategy was not toxic to the sperm; it did not kill them or reduce their motility, nor did it affect their fertilization capacity. However, we did observe developmental delays among pre-implantation embryos created using PFAS-treated sperm. Such findings raise the intriguing prospect that PFAS-exposed sperm harbor a form of stress signal that they deliver to the embryo upon fertilization.

Direct In Vitro Comparison of the Anti-Leishmanial Activity of Different Olive Oil Total Polyphenolic Fractions and Assessment of Their Combined Effects with Miltefosine.

The bioactive compounds present in the edible products of the olive tree have been extensively studied and their favorable effects on various disease risk factors have been demonstrated. The aim of this study was to perform a comparative analysis of the anti-leishmanial effects of total phenolic fractions (TPFs) derived from extra virgin olive oil with different phenolic contents and diverse quantitative patterns. Moreover, the present study investigated their association with miltefosine, a standard anti-leishmanial drug, against both extracellular promastigotes and intracellular amastigotes of a viscerotropic and a dermotropic Leishmania strain. The chemical compositions of TPFs were determined by high performance liquid chromatography with diode array detection (HPLC-DAD). Analysis of parasite growth kinetics, reactive oxygen species production and apoptotic events were determined by microscopy and flow cytometry. Our results revealed that the presence of oleacein (OLEA) and oleocanthal (OLEO) secoiridoids enhances the anti-leishmanial effect of TPF. The association between TPFs and miltefosine was suggested as being additive in Leishmania infantum and Leishmania major promastigotes, and as antagonistic in intracellular amastigotes, as was evaluated with the modified isobologram method. The obtained data verified that TPFs are bioactive dietary extracts with a strong anti-leishmanial activity and highlighted that fractions that are richer in OLEA and OLEO phenolic compounds possess stronger inhibitory effects against parasites. This study may contribute to improving the therapeutic approaches against leishmaniasis.

Skin Toxicity Assessment of Silver Nanoparticles in a 3D Epidermal Model Compared to 2D Keratinocytes.

INTRODUCTION: Increased use of silver nanoparticles (AgNPs) has raised concerns that AgNPs may induce toxic effects. In vitro studies of cell monolayers and in vivo studies have produced conflicting results. The inconsistency of these results has been mainly due to limitations of two-dimensional (2D) monolayer cell systems. METHODS: A three-dimensional (3D) epidermal model called EpiKutis®, which exhibits good tissue viability and barrier function was developed. The cytotoxicity of AgNPs against EpiKutis was compared to that against 2D keratinocytes at equivalent AgNPs doses (0.035, 0.07, 0.14, 0.28, and 0.56 ng per cell). The amount and distribution of AgNPs in the 3D EpiKutis and 2D keratinocytes after exposure were determined. The toxic mechanisms of AgNPs, such as oxidative stress and production of pro-inflammatory cytokines, were investigated. RESULTS: The results demonstrated that cell viability was greater than 80% and lactate dehydrogenase (LDH) release did not increase even at the highest dose of AgNPs in EpiKutis. In contrast, treatment of 2D keratinocytes with AgNPs resulted in dose-dependent decrease in cell viability from 63% to 11%, and a dose-dependent increase in LDH release from 8% to 16%. Cytotoxicity of AgNPs in 2D keratinocytes was related to oxidative damage and inflammation, as evidenced by increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), IL-1α, IL-6, and IL-8. In addition, levels of superoxide dismutase (SOD) were decreased. EpiKutis treated with AgNPs did not exhibit increased oxidative damage or inflammation, which may have been due to the barrier properties of the 3D structure, resulting in reduced penetration of AgNPs. At equivalent per cell doses, total silver penetration into EpiKutis was 0.9 ± 0.1%, and total silver penetration into 2D keratinocytes was 8.8 ± 0.6% detected by ICP-MS. The penetration and distribution of AgNPs in 2D keratinocytes were confirmed by the TEM-EDS analysis, which was not found in the 3D EpiKutis. These results showed that AgNPs penetrated EpiKutis to a lesser degree than they penetrated 2D keratinocytes, which suggested that EpiKutis exhibited significant barrier function. DISCUSSION: The results of this study showed that AgNP toxicity should be evaluated using 3D epidermal models, which may provide better estimates of in vivo conditions than 2D models. The EpiKutis model may be an ideal model for assessment of nanotoxicity.