Assessing the impact of benzo[a]pyrene with the in vitro fish gut model: An integrated approach for eco-genotoxicological studies.

In vitro models are emerging tools for reducing reliance on traditional toxicity tests, especially in areas where information is sparse. For studies of fish, this is especially important for extrahepatic organs, such as the intestine, which, until recently, have been largely overlooked in favour of the liver or gill. Considering the importance of dietary uptake of contaminants, the rainbow trout (Oncorhynchus mykiss) intestine-derived cell line RTgutGC was cultured, to test its suitability as a high-throughput in vitro model. Benzo[a]pyrene (B[a]P) is an important contaminant and a model polycyclic aromatic hydrocarbon (PAH). Over 48 h exposure, a range of endpoints and xenobiotic metabolism rates were examined at three different pH levels indicative of the in vitro (pH 7.5) and in vivo mid-gut (pH 7.7) and hind-gut (pH 7.4) regions as a function of time. These endpoints included (i) cell viability: acid phosphatase (APH) and lactate dehydrogenase (LDH) assays; (ii) glucose uptake; (iii) cytochrome P450 enzyme activity: 7-ethoxyresoorufin-O-deethylase (EROD) assay; (iv) glutathione transferase (GST) activity; (v) genotoxic damage determined using the comet assay. Absence of cell viability loss, in parallel with decrease in the parent compound (B[a]P) in the medium and its subsequent increase in the cells suggested active sequestration, biotransformation, and removal of this representative PAH. With respect to genotoxic response, significant differences were observed at both the sampling times and the two highest concentrations of B[a]P. No significant differences were observed for the different pH conditions. Overall, this in vitro xenobiotic metabolism system appears to be a robust model, providing a basis for further development to evaluate metabolic and toxicological potential of contaminants without use of animals

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

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.]

ACSM Expert Consensus Statement on Weight Loss in Weight-Category Sports

Weight-category sports are defined by the requirement of a weigh-in before competition to provide performance equity and reduced injury risks by eliminating size discrepancies. Athletes in these sports try to gain a theoretical advantage by competing in weight divisions that are lower than their day-to-day body mass (BM), using a combination of chronic strategies (body-fat losses) and acute manipulations over a period of hours to days before weigh-in ("making weight"). Strategies to support safer practices include minimal competition weight classification based on preseason body composition, reductions in the period between weigh-in and competition, and prohibition of unhealthy weight loss techniques. At an individual level, expert guidance by a sports nutrition professional can help an athlete to establish a pragmatic and long-term approach to BM management, recognizing the nuances of their sport, to achieve favorable outcomes for both health and performance

Multimorbidity in bipolar disorder and under-treatment of cardiovascular disease: a cross sectional study

Background: Individuals with serious mental disorders experience poor physical health, especially increased rates of cardiometabolic morbidity and premature morbidity. Recent evidence suggests that individuals with schizophrenia have numerous comorbid physical conditions which may be under-recorded and under-treated but to date very few studies have explored this issue for bipolar disorder. Methods:We conducted a cross-sectional analysis of a dataset of 1,751,841 registered patients within 314 primary-care practices in Scotland, U.K. Bipolar disorder was identified using Read Codes recorded within electronic medical records. Data on 32 common chronic physical conditions were also assessed. Potential prescribing inequalities were evaluated by analyzing prescribing data for coronary heart disease (CHD) and hypertension. Results: Compared to controls, individuals with bipolar disorder were significantly less likely to have no recorded physical conditions (OR 0.59, 95% CI 0.54-0.63) and significantly more likely to have one physical condition (OR 1.27, 95% CI 1.16-1.39), two physical conditions (OR 1.45, 95% CI 1.30-1.62) and three or more physical conditions (OR 1.44, 95% CI 1.30-1.64). People with bipolar disorder also had higher rates of thyroid disorders, chronic kidney disease, chronic pain, chronic obstructive airways disease and diabetes but, surprisingly, lower recorded rates of hypertension and atrial fibrillation. People with bipolar disorder and comorbid CHD or hypertension were significantly more likely to be prescribed no antihypertensive or cholesterol-lowering medications compared to controls, and bipolar individuals with CHD or hypertension were significantly less likely to be on 2 or more antihypertensive agents. Conclusions: Individuals with bipolar disorder are similar to individuals with schizophrenia in having a wide range of comorbid and multiple physical health conditions. They are also less likely than controls to have a primary-care record of cardiovascular conditions such as hypertension and atrial fibrillation. Those with a recorded diagnosis of CHD or hypertension were less likely to be treated with cardiovascular medications and were treated less intensively. This study highlights the high physical healthcare needs of people with bipolar disorder, and provides evidence for a systematic under-recognition and under-treatment of cardiovascular disease in this group

Hormonal contraceptive use, menstrual cycle characteristics and training/nutrition related profiles of elite, sub-elite and amateur athletes and exercisers: One size is unlikely to fit all

The aim of the present study was to simultaneously audit hormonal contraceptive (HC) use, menstrual cycle characteristics and training/nutrition related profiles in pre-menopausal women from varying athletic and exercise backgrounds. Elite (n = 51), sub-elite (n = 118) and amateur (n = 392) female athletes and exercisers were examined via an anonymous quantitative/qualitative survey tool. All analyses for ratio data were conducted utilising one- and two-way ANOVA/ANCOVA and odds ratio models, with ordinal data analysed via Pearson's Chi-squared tests. HC use was similar across elite, sub-elite and amateurs (34–44%). Menstrual cycle length was not different (P = 0.08) between competitive levels (28 ± 13 days), but 66% of respondents reported cycle variability (10 ± 11 days). Training profiles were not different based on contraceptive status (P > 0.05) yet were across competitive groups (P 0.05), though elite and sub-elite groups reported a higher daily meal consumption compared to amateurs (P < 0.01). Forty percent of all respondents skipped meals, attributed to lack of time, schedule and alterations in appetite, with reported changes in taste preferences during pre-menses (14–35%) and menses (15–25%) towards sweet foods (60%), with cravings for chocolate and other confectionary (25%). Prevalence of dietary supplement use was not influenced by contraceptive status (P = 0.31), though elites (76%) reported higher use (P = 0.04) than amateurs (63%). Data demonstrates that in female athletes and exercisers, competitive level appears to affect simple markers of training and nutrition practice, yet contraceptive status does not

Regional Chemotherapy in Locally Advanced Pancreatic Cancer: RECLAP Trial

<p>Abstract</p> <p>Background</p> <p>Pancreatic cancer is the fourth leading cause of cancer death in the United States. Surgery offers the only chance for cure. However, less than twenty percent of patients are considered operative candidates at the time of diagnosis. A common reason for being classified as unresectable is advanced loco-regional disease.</p> <p>A review of the literature indicates that almost nine hundred patients with pancreatic cancer have received regional chemotherapy in the last 15 years. Phase I studies have shown regional administration of chemotherapy to be safe. The average reported response rate was approximately 26%. The average 1-year survival was 39%, with an average median survival of 9 months. Of the patients that experienced a radiographic response to therapy, 78 (78/277, 28%) patients underwent exploratory surgery following regional chemotherapy administration; thirty-two (41%) of those patients were amenable to pancreatectomy. None of the studies performed analyses to identify factors predicting response to regional chemotherapy.</p> <p>Progressive surgical techniques combined with current neoadjuvant chemoradiotherapy strategies have already yielded emerging support for a multimodality approach to treatment of advanced pancreatic cancer.</p> <p>Intravenous gemcitabine is the current standard treatment of pancreatic cancer. However, >90% of the drug is secreted unchanged affecting toxicity but not the cancer per se. Gemcitabine is converted inside the cell into its active drug form in a rate limiting reaction. We hypothesize that neoadjuvant regional chemotherapy with continuous infusion of gemcitabine will be well tolerated and may improve resectability rates in cases of locally advanced pancreatic cancer.</p> <p>Design</p> <p>This is a phase I study designed to evaluate the feasibility and toxicity of super-selective intra-arterial administration of gemcitabine in patients with locally advanced, unresectable pancreatic adenocarcinoma. Patients considered unresectable due to locally advanced pancreatic cancer will receive super-selective arterial infusion of gemcitabine over 24 hours via subcutaneous indwelling port. Three to six patients will be enrolled per dose cohort, with seven cohorts, plus an additional six patients at the maximum tolerated dose; accrual is expected to last 36 months. Secondary objectives will include the determination of progression free and overall survival, as well as the conversion rate from unresectable to potentially resectable pancreatic cancer.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov ID: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01294358">NCT01294358</a></p

Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper.

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the "gut sac" method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner

Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance

At high internal doses, pharmaceuticals have the potential for inducing biological/pharmacological effects in fish. One particular concern for the environment is their potential to bioaccumulate and reach pharmacological levels; the study of these implications for environmental risk assessment has therefore gained increasing attention. To avoid unnecessary testing on animals, in vitro methods for assessment of xenobiotic metabolism could aid in the ecotoxicological evaluation. Here we report the use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout to measure the metabolism of seven pharmaceuticals using a substrate depletion assay. Of the pharmaceuticals tested, propranolol, diclofenac and phenylbutazone were metabolised by trout liver spheroids; atenolol, metoprolol, diazepam and carbamazepine were not. Substrate depletion kinetics data was used to estimate intrinsic hepatic clearance by this spheroid model, which was similar for diclofenac and approximately 5 fold higher for propranolol when compared to trout liver microsomal fraction (S9) data. These results suggest that liver spheroids could be used as a relevant and metabolically competent in vitro model with which to measure the biotransformation of pharmaceuticals in fish; and propranolol acts as a reproducible positive control