Spheroid Size Does not Impact Metabolism of the ß-blocker Propranolol in 3D Intestinal Fish Model.

Compared to two-dimensional (2D) cell culture, cellular aggregates or spheroids (3D) offer a more appropriate alternative in vitro system where individual cell-cell communication and micro-environment more closely represent the in vivo organ; yet we understand little of the physiological conditions at this scale. The relationship between spheroid size and oxygen microenvironment, an important factor influencing the metabolic capacity of cells, was first established using the fish intestine derived RTgutGC cell line. Subsequently, pharmaceutical metabolism (Propranolol), as determined by high performance liquid chromatography, in this intestinal model was examined as a function of spheroid size. Co-efficient of variation between spheroid size was below 12% using the gyratory platform method, with the least variation observed in the highest cell seeding density. The viable, high oxygen micro-environment of the outer rim of the spheroid, as determined by electron paramagnetic resonance (EPR) oximetry, decreased over time, and the hypoxic zone increased as a function of spheroid size. Despite a trend of higher metabolism in smaller spheroids, the formation of micro-environments (quiescent, hypoxic or anoxic) did not significantly affect metabolism or function of an environmentally relevant pharmaceutical in this spheroid model.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia.

In vivo, mammalian cells reside in an environment of 0.5-10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

Linking genotoxicity and cytotoxicity with membrane fluidity: A comparative study in ovarian cancer cell lines following exposure to auranofin.

Auranofin, an organogold compound classified as an anti-rheumatic agent is under phase 2 clinical trials for re-purposing to treat recurrent epithelial ovarian cancer. We have reported earlier that Breast cancer 1, early onset (BRCA1) mutant ovarian cancer cells exhibit increased sensitivity to auranofin. BRCA1 is a DNA repair protein whose functional status is critical in the prognosis of ovarian cancer. Apart from DNA repair capability of cancer cells, membrane fluidity is also implicated in modulating resistance to chemotherapeutics. We report here that membrane fluidity influences the sensitivity of ovarian cancer cell lines, OVCAR5 and IGROV1, to auranofin. Electron spin resonance (ESR) analysis revealed a more fluidized membrane in IGROV1 compared to OVCAR5. Interestingly, IGROV1 cells were more sensitive to auranofin induced cytotoxicity than OVCAR5. In comparison to OVCAR5, IGROV1 cells also exhibited an increased number of DNA double strand breaks (DSBs) upon auranofin treatment as assessed by 53BP1 immunostaining. Furthermore, correlation analysis demonstrated a strong positive correlation (r=0.856) between membrane fluidity and auranofin sensitivity in these cell lines. Auranofin-treated IGROV1 cells also exhibited increased cellular oxidation and apoptosis. Anti-oxidant, N-acetyl cysteine (NAC) inhibited the cellular oxidation and apoptosis in auranofin-treated ovarian cancer cells suggesting reactive oxygen species (ROS) mediate the anti-cancer properties of auranofin. Overall, our study suggests that auranofin mediates its cytotoxicity via ROS production in ovarian cancer cells which correlates positively with membrane fluidity.

Correction: Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry.

[This corrects the article DOI: 10.1371/journal.pone.0149492.].

The hydroxypyridinone iron chelator CP94 increases methyl-aminolevulinate-based photodynamic cell killing by increasing the generation of reactive oxygen species.

Methyl-aminolevulinate-based photodynamic therapy (MAL-PDT) is utilised clinically for the treatment of non-melanoma skin cancers and pre-cancers and the hydroxypyridinone iron chelator, CP94, has successfully been demonstrated to increase MAL-PDT efficacy in an initial clinical pilot study. However, the biochemical and photochemical processes leading to CP94-enhanced photodynamic cell death, beyond the well-documented increases in accumulation of the photosensitiser protoporphyrin IX (PpIX), have not yet been fully elucidated. This investigation demonstrated that MAL-based photodynamic cell killing of cultured human squamous carcinoma cells (A431) occurred in a predominantly necrotic manner following the generation of singlet oxygen and ROS. Augmenting MAL-based photodynamic cell killing with CP94 co-treatment resulted in increased PpIX accumulation, MitoSOX-detectable ROS generation (probably of mitochondrial origin) and necrotic cell death, but did not affect singlet oxygen generation. We also report (to our knowledge, for the first time) the detection of intracellular PpIX-generated singlet oxygen in whole cells via electron paramagnetic resonance spectroscopy in conjunction with a spin trap.

Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry.

Advanced in vitro culture from tissues of different origin includes three-dimensional (3D) organoid micro structures that may mimic conditions in vivo. One example of simple 3D culture is spheroids; ball shaped structures typically used as liver and tumour models. Oxygen is critically important in physiological processes, but is difficult to quantify in 3D culture: and the question arises, how small does a spheroid have to be to have minimal micro-environment formation? This question is of particular importance in the growing field of 3D based models for toxicological assessment. Here, we describe a simple non-invasive approach modified for the quantitative measurement and subsequent evaluation of oxygen gradients in spheroids developed from a non-malignant fish cell line (i.e. RTG-2 cells) using Electron Paramagnetic Resonance (EPR) oximetry. Sonication of the paramagnetic probe Lithium phthalocyanine (LiPc) allows for incorporation of probe particulates into spheroid during its formation. Spectra signal strength after incorporation of probe into spheroid indicated that a volume of 20 µl of probe (stock solution: 0.10 mg/mL) is sufficient to provide a strong spectra across a range of spheroid sizes. The addition of non-toxic probes (that do not produce or consume oxygen) report on oxygen diffusion throughout the spheroid as a function of size. We provide evidence supporting the use of this model over a range of initial cell seeding densities and spheroid sizes with the production of oxygen distribution as a function of these parameters. In our spheroid model, lower cell seeding densities (∼2,500 cells/spheroid) and absolute size (118±32 µm) allow control of factors such as pre-existing stresses (e.g. ∼ 2% normoxic/hypoxic interface) for more accurate measurement of treatment response. The applied methodology provides an elegant, widely applicable approach to directly characterize spheroid (and other organoid) cultures in biomedical and toxicological research.

Photoexcitation of aqueous suspensions of titanium dioxide nanoparticles: an electron spin resonance spin trapping study of potentially oxidative reactions.

It is well-established that exposure of aqueous suspensions of titanium dioxide (TiO(2)) nanoparticles to ultraviolet A (UVA) light produces reactive oxygen species which leads to biological damage. However, there is disagreement in the literature as to the exact nature of these species and how they are formed. Using a number of different spin traps (i.e. PBN, POBN, DMPO, DEPMPO), we have shown that the primary damaging species produced on irradiation of an aqueous suspension of TiO(2) is the hydroxyl radical, which is formed at the valence band hole under both aerobic and hypoxic conditions. Hydroxyl radical production is enhanced by the presence of oxygen which probably reacts with the conduction band electrons or resultant Ti(3+), inhibiting hole-electron recombination, although we find no evidence of reaction of oxygen to form free superoxide radical anions or of the formation of any other radical at that site. The present results suggest that the resulting O(2)(•-) species may not be as labile as previously thought and may possibly undergo further reduction to the O(2)(2-) dianion. Hydroxyl radicals formed at the surface of the TiO(2) readily react with substrates containing an abstractable hydrogen to produce secondary radicals that, in biological systems, could lead to cell damage.

Titanium dioxide induced cell damage: a proposed role of the carboxyl radical.

Titanium dioxide (TiO(2)) nanoparticles have been shown to be genotoxic to cells exposed to ultraviolet A (UVA) radiation. Using the technique of electron spin resonance (ESR) spin trapping, we have confirmed that the primary damaging species produced on irradiation of TiO(2) nanoparticles is the hydroxyl (OH) radical. We have applied this technique to TiO(2)-treated fish and mammalian cells under in vitro conditions and observed the additional formation of carboxyl radical anions (CO(2)(-)) and superoxide radical anions (O(2)(-)). This novel finding suggests a hitherto unreported pathway for damage, involving primary generation of OH radicals in the cytoplasm, which react to give CO(2)(-) radicals. The latter may then react with cellular oxygen to form O(2)(-) and genotoxic hydrogen peroxide (H(2)O(2)).

Hydroxyl radicals (*OH) are associated with titanium dioxide (TiO(2)) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells.

TiO(2) nanoparticles (< 100 nm diameter) have been reported to cause oxidative stress related effects, including inflammation, cytotoxicity and genomic instability, either alone or in the presence of UVA irradiation in mammalian studies. Despite the fact that the aquatic environment is often the ultimate recipient of all contaminants there is a paucity of data pertaining to the potential detrimental effects of nanoparticles on aquatic organisms. Therefore, these investigations aimed to evaluate the potential cytotoxic and genotoxic effects of TiO(2) nanoparticles on goldfish skin cells (GFSk-S1), either alone or in combination with UVA. Whilst neutral red retention (NRR) assay (a measure of lysosomal membrane integrity) was used to evaluate cell viability, a modified Comet assay using bacterial lesion-specific repair endonucleases (Endo-III, Fpg) was employed to specifically target oxidative DNA damage. Additionally, electron spin resonance (ESR) studies with different spin traps were carried out for qualitative analysis of free radical generation. For cell viability, TiO(2) alone (0.1-1000 microg ml(-1)) had little effect whereas co-exposure with UVA (0.5-2.0 kJm(-2)) caused a significant dose-dependent decrease which was dependent on both the concentration of TiO(2) and the dose of UVA administered. For the Comet assay, doses of 1, 10 and 100 microg ml(-1) in the absence of UVA caused elevated levels of Fpg-sensitive sites, indicating the oxidation of purine DNA bases (i.e. guanine) by TiO(2). UVA irradiation of TiO(2)-treated cells caused further increases in DNA damage. ESR studies revealed that the observed toxic effects of nanoparticulate TiO(2) were most likely due to hydroxyl radical (OH) formation.