Comparative effects of TiO2 and ZnO nanoparticles on growth and ultrastructure of ovarian antral follicles.

Titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NP) have been demonstrated to reach the ovary. However, the potential detrimental effects of these metal-based NP on ovarian antral follicles and whether they can be directly taken up by follicular cells are unknown. The aim of this study was to evaluate whether TiO2 and ZnO NP internalize into the antral follicle, and further compared any potential detrimental effects of either NP on growth, ultrastructure and viability of antral follicles. It has been described that TiO2 and ZnO NP induce oxidative stress, thus this study indirectly assessed whether oxidative stress was involved. Antral follicles were cultured with TiO2 (5, 25 and 50 µg/mL) or ZnO (5, 15 and 25 µg/mL) NP for 96 h. TiO2 NP were internalized and agglomerated into cells, increased follicle diameter and disrupted the cytoskeleton arrangement, effects that were partially prevented by a co-exposure with trolox. Moreover, ZnO NP partially dissolved into culture media, decreased follicle diameter, and disrupted cytoskeletal arrangement, and these effects were not prevented by trolox. Ultrastructural alterations induced by exposure to both NP were evidenced by impaired transzonal projections and swelling mitochondria. Oxidative stress mediates TiO2 NP-induced effects but not those from ZnO NP in antral follicle development. Our results suggest that both NP induced ovarian follicle toxicity through different toxic mechanisms, possibly due to a stimulation of ZnO NP solubility and agglomeration of TiO2 NP into the follicular cells.

Bisphenol A alters oocyte maturation by prematurely closing gap junctions in the cumulus cell-oocyte complex.

In ovarian follicles, cumulus cells communicate with the oocyte through gap junction intercellular communication (GJIC), to nurture the oocyte and control its meiosis arrest and division. Bisphenol A (BPA) is a monomer found in polycarbonate-made containers that can induce functional alterations, including impaired oocyte meiotic division and reduced molecule transfer in GJIC. However, how BPA alters oocyte meiotic division is unclear. We investigated whether BPA effects on oocyte meiotic division were correlated with reduced transfer in GJIC. Cumulus cell-oocyte complexes (COCs) isolated from mouse preovulatory follicles were cultured with 0, 0.22, 2.2, 22, 220, and 2200 nM BPA for 2 h. An additional 16-h incubation with epidermal growth factor (EGF) was performed to promote the occurrence of meiotic resumption and progression to metaphase II. Without EGF stimulus, BPA treatment increased the percentage of oocytes undergoing meiotic resumption, decreased GJIC in the COCs, and did not modify GJIC gene (Cx43 and Cx37) and protein (CX43) expression. Following EGF stimulus, BPA increased the percentage of oocytes that remained at the anaphase and telophase stages, and decreased the percentage of oocytes reaching the metaphase II stage. Concomitantly, BPA reduced the expansion of cumulus cells. Carbenoxolone (a GJIC inhibitor) and 6-diazo-5-oxo-l-norleucine (a cumulus cell-expansion inhibitor) exerted effects on meiotic division similar to those exerted by BPA. These data suggest that BPA accelerates meiotic progression, leading to impaired prophase I-to-metaphase II transition, and that this adverse effect is correlated with reduced bidirectional communication in the COC.

Resveratrol induces cell death in cervical cancer cells through apoptosis and autophagy.

Cervical neoplasia is one of the most frequent cancers in women and is associated with high-risk human papillomavirus (HPV) infection. Resveratrol, a natural polyphenolic phytochemical, has received considerable interest on the basis of its potential as a chemopreventive agent against human cancer. In this work, we analyzed the type of cell death induced by resveratrol in several cervical cancer cell lines. Resveratrol treatment (150-250 µmol/l) for 48 h increased cell cycle arrest at the G1 phase in C33A (with mutation in p53) and HeLa cells (HPV18 positive), as well as in CaSki and SiHa cell lines (HPV16 positive). Resveratrol treatment induced apoptosis in all cell lines, particularly in CaSki cells, as measured by Annexin-V flow cytometry analysis. There was a decrease in the mitochondrial membrane potential (apoptosis) in HeLa, CaSki, and SiHa cells and an increased lysosomal permeability (autophagy) in C33A, CaLo (HPV18 positive), and HeLa cell lines. Furthermore, when we used the IC50 of each line, we found that resveratrol produces a similar effect, suggesting that this effect is not dependent on the concentration of resveratrol. Interestingly, after resveratrol treatment, the expression of p53 was decreased in HPV18-positive cell lines (CaLo and HeLa) and increased in HPV16-positive cell lines (CaSki and SiHa) and C33A cells. The expression of p65 (an NF-κB subunit) was decreased after treatment in all cell lines except SiHa cells. These data indicate that resveratrol uses different mechanisms to induce cell death in cell lines derived from cervical cancer.

Levocetirizine inhibits migration of immune cells to lymph nodes and induces treg cells in a murine type I allergic conjunctivitis model.

UNLABELLED: BACKGROUND #ENTITYSTARTX00026; PURPOSE: Levocetirizine is a histamine H(1) receptor antagonist. Here, we utilised DO11.10TCR transgenic mice to establish an antigen-specific T cell-dependent allergic conjunctivitis (AC) model to determine the effect of the topical application of an ophthalmic formulation of Levoceritizine as a treatment for AC. EXPERIMENTAL APPROACH: DO11.10 mice (n=6/each) were exposed to ovalbumin (OVA, 50 µg) and treated with a Levocetirizine ophthalmic formulation (0.001-0.02% v/w) or placebo (vehicle) for 24-72 h. Serum, aqueous/vitreous humour and conjunctiva were obtained. Immunoglobulin (Ig)-E, interleukin (IL)-10 and lipoxin (LX)A(4) were determined by ELISA. Levels of tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-ß, interferon (IFN)-γ and 18rS expression were measured by RT-PCR. Proportions of total and activated antigen-presenting cells (APC), recruited T lymphocytes (CD4+), activated T lymphocytes (CD25+) and T regulatory cells (Treg) were measured by flow cytometry. KEY RESULTS: OVA exposure induced AC in the animal model indicated by increased expression of LXA(4), TNF-α and TGF-ß. Levocetirizine treatment (0.01-0.02% v/w) reduced LXA(4) in the eye humours. This treatment approach increased systemic IL-10 secretion and reduced TNF-α and TGF-ß expression in conjunctiva without changing IFN-γ expression. Levocetirizine reduced APC levels in draining lymph nodes but increased the proportion of total lymphocytes recruited and their differentiation to Treg cells. CONCLUSIONS #ENTITYSTARTX00026; IMPLICATIONS: Levocetirizine effectively reduces the activation and migration of APC to local draining lymph nodes and induces differentiation of Treg cells as one possible mechanism of its anti-inflammatory action.