The effect of insulin resistance and exercise on the percentage of CD16(+) monocyte subset in obese individuals.

UNLABELLED: Obesity is a low-grade chronic inflammation condition, and macrophages, and possibly monocytes, are involved in the pathological outcomes of obesity. Physical exercise is a low-cost strategy to prevent and treat obesity, probably because of its anti-inflammatory action. We evaluated the percentage of CD16(-) and CD16(+) monocyte subsets in obese insulin-resistant individuals and the effect of an exercise bout on the percentage of these cells. Twenty-seven volunteers were divided into three experimental groups: lean insulin sensitive, obese insulin sensitive and obese insulin resistant. Venous blood samples collected before and 1 h after an aerobic exercise session on a cycle ergometer were used for determination of monocyte subsets by flow cytometry. Insulin-resistant obese individuals have a higher percentage of CD16(+) monocytes (14.8 ± 2.4%) than the lean group (10.0 ± 1.3%). A positive correlation of the percentage of CD16(+) monocytes with body mass index and fasting plasma insulin levels was found. One bout of moderate exercise reduced the percentage of CD16(+) monocytes by 10% in all the groups evaluated. Also, the absolute monocyte count, as well as all other leukocyte populations, in lean and obese individuals, increased after exercise. This fact may partially account for the observed reduction in the percentage of CD16(+) cells in response to exercise. Insulin-resistant, but not insulin-sensitive obese individuals, have an increased percentage of CD16(+) monocytes that can be slightly modulated by a single bout of moderate aerobic exercise. These findings may be clinically relevant to the population studied, considering the involvement of CD16(+) monocytes in the pathophysiology of obesity. Copyright © 2016 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDY: Obesity is now considered to be an inflammatory condition associated with many pathological consequences, including insulin resistance. It is proposed that insulin resistance contributes to the aggravation of the inflammatory dysfunction in obesity. The effect of obesity on the percentage of monocytes was previously observed in class II and III obese individuals who presented other alterations in addition to insulin resistance. In this study we observed that insulin-resistant obese individuals, but not insulin-sensitive ones, had an increased percentage of CD14(+) CD16(+) monocytes. This fact shows that a dysfunction of the monocyte percentage in class I obese individuals is only seen when this condition is associated with insulin resistance.

Impact of creatine on muscle performance and phosphagen stores after immobilization.

PURPOSE: This study investigated the effect of creatine (CR) supplementation during cast-immobilization to preserve skeletal muscle total work, power and intramuscular phosphocreatine (PCr) kinetics during dynamic exercise. METHODS: Twenty-five active individuals (24 ± 4 years,) performed wrist flexion exercise within a 1.9 Tesla superconducting magnet before and after 1 week of cast-immobilization. An incremental protocol to fatigue and two constant load (CL1 and CL2) exercise bouts were performed. While casted, participants consumed either 20 g day(-1) of CR or a placebo (PLA). (31)P magnetic resonance spectroscopy was used to quantify in vivo intramuscular PCr levels. RESULTS: No significant group × time interaction effects were found for work or power throughout all exercise bouts. Total work was significantly reduced over time in both groups (p = 0.049) during the incremental exercise bout. Work production in CL1 tended (p = 0.073) to attenuate in the CR group, compared to PLA. No changes were observed in CL2. Baseline PCr significantly decreased with casting in PLA (PRE: 26.6 ± 6.3 vs. POST: 22.5 ± 5.6 mM kg(-1) wet muscle, p = 0.003). No change (p = 0.31) was observed in the CR group. Changes in work production were significantly correlated with changes in resting PCr in CR (r = -0.63, p = 0.021) but not PLA (r = -0.36, p = 0.26) group. CONCLUSIONS: Results suggest decreases in short-term endurance may be due to alternations of PCr status and/or metabolism. More research is needed to fully determine the efficacy of CR supplementation during short-term immobilization.

Palm Cooling and Heating Delays Fatigue During Resistance Exercise in Women.

We previously reported that cold application to the palms between sets of high-intensity bench press exercise produces an ergogenic effect in men. In this study, we hypothesized that palm cooling (PC) or heating during rest intervals between high-intensity weight training sets will increase total repetitions and exercise volume load (kilograms) in resistance trained female subjects in a thermoneutral (TN) environment. Eight female subjects (mean ± SD, age = 25 ± 6 years, height = 160 ± 6 cm, body mass = 56 ± 7 kg, 1-repetition maximum [1RM] = 52 ± 6 kg, weight training experience = 6 ± 2 years) completed 4 sets of 85% 1RM bench press exercise to failure, with 3-minute rest intervals. Exercise trials were performed in a counterbalanced order on 3 days, separated by at least 3 days in TN, Palm heating (PH), and PC conditions. Heating and cooling were applied by placing both hands in a hand cooling device with the hand plate set to 45° C for heating and 10° C for cooling. Data were analyzed using a 2-factor repeated-measures analysis of variance and Tukey's post hoc tests. Palm cooling repetitions were significantly higher than TN repetitions during the second set, and PH repetitions were significantly higher than those of TN during the fourth set. Total exercise volume load (kilograms) for both PC (1,387 ± 358) and PH (1,349 ± 267) were significantly higher than TN (1,187 ± 262). In women, both heating and cooling of the palms between sets of resistance exercise increased the total exercise volume load performed. This ergogenic response to a peripheral sensory input is consistent with the central governor theory of muscular fatigue.

Regulatory coordination between two major intracellular homeostatic systems: heat shock response and autophagy.

The eukaryotic cell depends on multitiered homeostatic systems ensuring maintenance of proteostasis, organellar integrity, function and turnover, and overall cellular viability. At the two opposite ends of the homeostatic system spectrum are heat shock response and autophagy. Here, we tested whether there are interactions between these homeostatic systems, one universally operational in all prokaryotic and eukaryotic cells, and the other one (autophagy) is limited to eukaryotes. We found that heat shock response regulates autophagy. The interaction between the two systems was demonstrated by testing the role of HSF-1, the central regulator of heat shock gene expression. Knockdown of HSF-1 increased the LC3 lipidation associated with formation of autophagosomal organelles, whereas depletion of HSF-1 potentiated both starvation- and rapamycin-induced autophagy. HSP70 expression but not expression of its ATPase mutant inhibited starvation or rapamycin-induced autophagy. We also show that exercise induces autophagy in humans. As predicted by our in vitro studies, glutamine supplementation as a conditioning stimulus prior to exercise significantly increased HSP70 protein expression and prevented the expected exercise induction of autophagy. Our data demonstrate for the first time that heat shock response, from the top of its regulatory cascade (HSF-1) down to the execution stages delivered by HSP70, controls autophagy thus connecting and coordinating the two extreme ends of the homeostatic systems in the eukaryotic cell.

Exercise regulation of intestinal tight junction proteins.

Gastrointestinal distress, such as diarrhoea, cramping, vomiting, nausea and gastric pain are common among athletes during training and competition. The mechanisms that cause these symptoms are not fully understood. The stress of heat and oxidative damage during exercise causes disruption to intestinal epithelial cell tight junction proteins resulting in increased permeability to luminal endotoxins. The endotoxin moves into the blood stream leading to a systemic immune response. Tight junction integrity is altered by the phosphoylation state of the proteins occludin and claudins, and may be regulated by the type of exercise performed. Prolonged exercise and high-intensity exercise lead to an increase in key phosphorylation enzymes that ultimately cause tight junction dysfunction, but the mechanisms are different. The purpose of this review is to (1) explain the function and physiology of tight junction regulation, (2) discuss the effects of prolonged and high-intensity exercise on tight junction permeability leading to gastrointestinal distress and (3) review agents that may increase or decrease tight junction integrity during exercise.

Historical control histopathology data from amphibian metamorphosis assays and fathead minnow fish short term reproductive assays: A tool for data interpretation.

The Amphibian Metamorphosis Assay (AMA) is used to determine if a tested chemical has potential to impact the hypothalamic-pituitary-thyroid (HPT) axis of Xenopus laevis tadpoles, while the Fish Short Term Reproduction Assay (FSTRA) assesses potential effects on the hypothalamic-pituitary-gonadal (HPG) axis of fish such as the fathead minnow (Pimephales promelas). Several global regulatory programs routinely require these internationally validated tests be performed to determine the potential endocrine activity of chemicals. As such, they are conducted in accordance with standardized protocols and test criteria, which were originally developed more than a decade ago. Sizeable numbers of AMA and FSTRA studies have since been carried out, which allows for the mining of extensive historical control data (HCD). Such data are useful for investigating the existence of outlier results and aberrant control groups, identifying potential confounding variables, providing context for rare diagnoses, discriminating target from non-target effects, and for refining current testing paradigms. The present paper provides histopathology HCD from 55 AMA studies and 45 fathead minnow FSTRA studies, so that these data may become publicly available and thus aid in the interpretation of future study outcomes. Histopathology is a key endpoint in these assays, in which it is considered to be one of the most sensitive indicators of endocrine perturbation. In the current review, granular explorations of HCD data were used to identify background lesions, to assess the utility of particular diagnostic findings for distinguishing endocrine from non-endocrine effects, and to help determine if specific improvements to established regulatory guidance may be warranted. Knowledge gleaned from this investigation, supplemented by information from other recent studies, provided further context for the interpretation of AMA and FSTRA histopathology results. We recommend HCDs for the AMA and FSTRA be maintained to support the interpretation of study results.

Space physiology VI: exercise, artificial gravity, and countermeasure development for prolonged space flight.

When applied individually, exercise countermeasures employed to date do not fully protect the cardiovascular and musculoskeletal systems during prolonged spaceflight. Recent ground-based research suggests that it is necessary to perform exercise countermeasures within some form of artificial gravity to prevent microgravity deconditioning. In this regard, it is important to provide normal foot-ward loading and intravascular hydrostatic-pressure gradients to maintain musculoskeletal and cardiovascular function. Aerobic exercise within a centrifuge restores cardiovascular function, while aerobic exercise within lower body negative pressure restores cardiovascular function and helps protect the musculoskeletal system. Resistive exercise with vibration stimulation may increase the effectiveness of resistive exercise by preserving muscle function, allowing lower intensity exercises, and possibly reducing risk of loss of vision during prolonged spaceflight. Inexpensive methods to induce artificial gravity alone (to counteract head-ward fluid shifts) and exercise during artificial gravity (for example, by short-arm centrifuge or exercise within lower body negative pressure) should be developed further and evaluated as multi-system countermeasures.

Effect of local cooling on short-term, intense exercise.

The widespread belief that local cooling impairs short-term, strenuous exercise performance is controversial. Eighteen original investigations involving cooling before and intermittent cooling during short-term, intensive exercise are summarized in this review. Previous literature examining short-term intensive exercise and local cooling primarily has been limited to the effects on muscle performance immediately or within minutes following cold application. Most previous cooling studies used equal and longer than 10 minutes of pre-cooling, and found that cooling reduced strength, performance and endurance. Because short duration, high intensity exercise requires adequate warm-up to prepare for optimal performance, prolonged pre-cooling is not an effective method to prepare for this type of exercise. The literature related to the effect of acute local cooling immediately before short duration, high intensity isotonic exercise such as weight lifting is limited. However, local intermittent cooling during short-term, high intense exercise may provide possible beneficial effects; first, by pain reduction, caused by an "irritation effect" from hand thermal receptors which block pain sensation, or second, by a cooling effect, whereby stimulation of hand thermal receptors or a slight lowering of blood temperature might alter central fatigue.

The Effect of Acidic and Alkaline Seawater on the F-Actin-Dependent Ca2+ Signals Following Insemination of Immature Starfish Oocytes and Mature Eggs.

In starfish, the addition of the hormone 1-methyladenine (1-MA) to immature oocytes (germinal vesicle, GV-stage) arrested at the prophase of the first meiotic division induces meiosis resumption (maturation), which makes the mature eggs able to respond to the sperm with a normal fertilization response. The optimal fertilizability achieved during the maturation process results from the exquisite structural reorganization of the actin cytoskeleton in the cortex and cytoplasm induced by the maturing hormone. In this report, we have investigated the influence of acidic and alkaline seawater on the structure of the cortical F-actin network of immature oocytes of the starfish (Astropecten aranciacus) and its dynamic changes upon insemination. The results have shown that the altered seawater pH strongly affected the sperm-induced Ca2+ response and the polyspermy rate. When immature starfish oocytes were stimulated with 1-MA in acidic or alkaline seawater, the maturation process displayed a strong dependency on pH in terms of the dynamic structural changes of the cortical F-actin. The resulting alteration of the actin cytoskeleton, in turn, affected the pattern of Ca2+ signals at fertilization and sperm penetration.

Helpful or harmful? The impact of the ketogenic diet on eating disorder outcomes in type 1 diabetes mellitus.

INTRODUCTION: Eating disorders (EDs) are common complications in people with type 1 diabetes (PwT1D), given the rigid focus on food and insulin dose adjustment. Dietary recommendations for T1D match those for the general population, yet many fail to achieve target HbA1c. Evidence suggests that lower carbohydrate meals and thus reduced insulin requirements may decrease inconsistencies in insulin absorption, maintain euglycemia and weight. Dietary restriction is a recognized risk factor for ED development, and Ketogenic Diets (KD) involve restriction of common family-based foods, thus impacting social normality and microbiome diversity. We reviewed the current literature on PwT1D following a KD to understand effects on ED risks. AREAS COVERED: Published data from MEDLINE, Embase, and PsycINFO were used. Search terms included: type 1 diabetes mellitus; or insulin dependent diabetes or T1D AND EDs or anorexia or bulimia or disordered eating AND low-carbohydrate diet or carbohydrate restricted diet or low carb diet or ketogenic diet. EXPERT OPINION: Research into the effects of KDs on ED outcomes in PwT1D are limited, given the concerns over risks of diabetic ketoacidosis, hypoglycemia, and dyslipidemia. Longer term studies on the participants' experience and motivations of adhering or admonishing the diet are needed.

Thermotolerance and heat acclimation may share a common mechanism in humans.

Thermotolerance and heat acclimation are key adaptation processes that have been hitherto viewed as separate phenomena. Here, we provide evidence that these processes may share a common basis, as both may potentially be governed by the heat shock response. We evaluated the effects of a heat shock response-inhibitor (quercetin; 2,000 mg/day) on established markers of thermotolerance [gastrointestinal barrier permeability, plasma TNF-α, IL-6, and IL-10 concentrations, and leukocyte heat shock protein 70 (HSP70) content]. Heat acclimation reduced body temperatures, heart rate, and physiological strain during exercise/heat stress) in male subjects (n = 8) completing a 7-day heat acclimation protocol. These same subjects completed an identical protocol under placebo supplementation (placebo). Gastrointestinal barrier permeability and TNF-α were increased on the 1st day of exercise/heat stress in quercetin; no differences in these variables were reported in placebo. Exercise HSP70 responses were increased, and plasma cytokines (IL-6, IL-10) were decreased on the 7th day of heat acclimation in placebo; with concomitant reductions in exercise body temperatures, heart rate, and physiological strain. In contrast, gastrointestinal barrier permeability remained elevated, HSP70 was not increased, and IL-6, IL-10, and exercise body temperatures were not reduced on the 7th day of heat acclimation in quercetin. While exercise heart rate and physiological strain were reduced in quercetin, this occurred later in exercise than with placebo. Consistent with the concept that thermotolerance and heat acclimation are related through the heat shock response, repeated exercise/heat stress increases cytoprotective HSP70 and reduces circulating cytokines, contributing to reductions in cellular and systemic markers of heat strain. Exercising under a heat shock response-inhibitor prevents both cellular and systemic heat adaptations.

A review of sex differences in immune function after aerobic exercise.

When menstrual phase and oral contraceptives are controlled for, males and females display marked differences in immune response to an exercise stress. In highly controlled research studies, sex differences in immune cell changes, cytokine alterations, along with morbidity and mortality after inoculation are apparent. Exercise has been hypothesized to serve as a model of various clinical stresses by inducing similar hormonal and immunological alterations. Thus, a greater understanding of sex differences in post exercise non-specific immune function may provide insight into more effective clinical approaches and treatments. This paper reviews the recent evidence supporting sex differences in post exercise immune response and highlights the need for greater control when comparing the post exercise immune response between sexes.

Effects of whole-body heat acclimation on cell injury and cytokine responses in peripheral blood mononuclear cells.

To test the hypothesis that whole-body heat acclimation (HA) would increase peripheral blood mononuclear cells' (PBMC) tolerance to heat shock (HS) and/or alter the release of cytokines (IL-1ß, IL-6, IL-10 and TNF-α) to bacterial lipopolysaccharide (LPS), we heat acclimated nine subjects by exercising them for 100 min in a hot environment for 10 days. The subjects' PBMC were separated and cultured on days 1 and 10 of HA pre- and post-exercise. Pre-exercise PBMC were allocated to three treatments: control (PRE, 37°C), HS (42.5°C for 2 h), or LPS (1 ng mL(-1) for 24 h). Post-exercise samples were incubated at 37°C. PBMC lactate dehydrogenase release increased (p < 0.05) after HS but it was not different (p > 0.05) between days 1 and 10 (0.100 ± 0.012 and 0.102 ± 0.16 abs., respectively). LPS treatment induced an increased (p < 0.05) release of cytokines but HA did not alter this response (p > 0.05). Pre-exercise intracellular heat shock protein 72 (Hsp72) was higher (p < 0.05) on day 10 compared to day 1 of HA (13 ± 5 and 8 ± 5 ng mL(-1), respectively). HS treatment caused a greater increase (p < 0.05) in Hsp72 than the exercise sessions on HA days 1 and 10. In addition, after HA, the Hsp72 response to HS was reduced (day 1, 129 ± 46; day 10, 80 ± 32 ng mL(-1), p < 0.05). In conclusion, HA increases PBMC Hsp72 but it does not reduce cellular damage to HS or alter cytokine response to LPS. We speculate that the stress applied during HA is not sufficient to modify the PBMC response.

Palm cooling to reduce heat strain in subjects during simulated armoured vehicle transport.

This study examined whether palm cooling (PC) could reduce heat strain, measured through changes in core, mean skin, mean body temperatures, and thermal sensation in resting hyperthermic subjects wearing chemical protective garments. Ten male subjects performed three exercise bouts (6.1 km h(-1), 2-4% grade) in a hot, dry environment [mean (SD) air temperature 42.2 (0.5 degrees C), relative humidity 36.5 (1%)] until core temperature reached 38.8 degrees C. Subjects then simulated transport in an armoured vehicle by resting in a seated position for 50 min with either no cooling (NC), (PC at 10 degrees C) or palm cooling with vacuum application around the hand (PCVAC, 10 degrees C, 7.47 kPa negative pressure). Core, skin, and mean body temperatures with PC and PCVAC were lower (P < 0.05) than NC from 15 to 50 min of cooling, and thermal sensation was lower (P < 0.05) from 30 to 50 min, with no differences in any variables between PC and PCVAC. Maximal heat extraction averaged 42 (12 W), and core temperature was reduced by 0.38 (0.21 degrees C) after 50 min of PC. Heat extraction with PC was modest compared to other cooling approaches in the literature.

Ibuprofen: Fish Short-Term Reproduction Assay with Zebrafish (Danio rerio) Based on an Extended OECD 229 Protocol.

A study was conducted to understand the potential for ibuprofen to impact the hypothalamus-pituitary-gonadal endocrine axis resulting in disruption of fish reproduction. The Good Laboratory Practice study was conducted according to the Organisation for Economic Co-operation and Development 229 Protocol, Fish Short-Term Reproduction Assay, and extended an additional 4 d to evaluate hatching success in the F1 generation. Test organisms were exposed to nominal test concentrations of 0.5, 2.4, 11.5, 55.3, and 265.4 µg ibuprofen/L and a negative control (dilution water). To strengthen the statistical power of the study, twice the number of replicates were used in the negative control versus individual treatment levels. A 21-d pre-exposure to identify groups of actively spawning fish was immediately followed by a 36-d exposure. Results for apical endpoints of survival, growth, and reproduction (fecundity and fertility), as well as the biomarker vitellogenin in the F0 generation and time to hatch and hatching success in the F1 generation are presented. Based on mean measured exposure concentrations and effects on fecundity in the F0 generation and hatching success in the F1 generation, overall no-observed-effect concentration and lowest-observed-effect concentration for the present study were 55.2 and 265.9 µg ibuprofen/L, respectively. Results from the present study indicate a lack of endocrine-mediated reproductive effects in zebrafish at environmentally relevant concentrations of ibuprofen. Environ Toxicol Chem 2020;39:1534-1545. © 2020 SETAC.

LBNP exercise protects aerobic capacity and sprint speed of female twins during 30 days of bed rest.

We have shown previously that treadmill exercise within lower body negative pressure (LBNPex) maintains upright exercise capacity (peak oxygen consumption, Vo(2peak)) in men after 5, 15, and 30 days of bed rest (BR). We hypothesized that LBNPex protects treadmill Vo(2peak) and sprint speed in women during a 30-day BR. Seven sets of female monozygous twins volunteered to participate. Within each twin set, one was randomly assigned to a control group (Con) and performed no countermeasures, and the other was assigned to an exercise group (Ex) and performed a 40-min interval (40-80% pre-BR Vo(2peak)) LBNPex (51 +/- 5 mmHg) protocol, plus 5 min of static LBNP, 6 days per week. Before and immediately after BR, subjects completed a 30.5-m sprint test and an upright graded treadmill test to volitional fatigue. These results in women were compared with previously reported reductions in Vo(2peak) and sprint speed in male twins after BR. In women, sprint speed (-8 +/- 2%) and Vo(2peak) (-6 +/- 2%) were not different after BR in the Ex group. In contrast, both sprint speed (-24 +/- 5%) and Vo(2peak) (-16 +/- 3%) were significantly less after BR in the Con group. The effect of BR on sprint speed and Vo(2peak) after BR was not different between women and men. We conclude that treadmill exercise within LBNP protects against BR-induced reductions in Vo(2peak) and sprint speed in women and should prove effective during long-duration spaceflight.

WISE-2005: effect of aerobic and resistive exercises on orthostatic tolerance during 60 days bed rest in women.

Cardiovascular deconditioning after long duration spaceflight is especially challenging in women who have a lower orthostatic tolerance (OT) compared with men. We hypothesized that an exercise prescription, combining supine aerobic treadmill exercise in a lower body negative pressure (LBNP) chamber followed by 10 min of resting LBNP, three to four times a week, and flywheel resistive training every third day would maintain orthostatic tolerance (OT) in women during a 60-day head-down-tilt bed rest (HDBR). Sixteen women were assigned to two groups (exercise, control). Pre and post HDBR OT was assessed with a tilt/LBNP test until presyncope. OT time (mean +/- SE) decreased from 17.5 +/- 1.0 min to 9.1 +/- 1.5 min (-50 +/- 6%) in control group (P < 0.001) and from 19.3 +/- 1.3 min to 13.0 +/- 1.9 min (-35 +/- 7%) in exercise group (P < 0.001), with no significant difference in OT time between the two groups after HDBR (P = 0.13). Nevertheless, compared with controls post HDBR, exercisers had a lower heart rate during supine rest (mean +/- SE, 71 +/- 3 vs. 85 +/- 4, P < 0.01), a slower increase in heart rate and a slower decrease in stroke volume over the course of tilt/LBNP test (P < 0.05). Blood volume (mean +/- SE) decreased in controls (-9 +/- 2%, P < 0.01) but was maintained in exercisers (-4 +/- 3%, P = 0.17).Our results suggest that the combined exercise countermeasure did not significantly improve OT but protected blood volume and cardiovascular response to sub tolerance levels of orthostatic stress.

Radical remedies: women, health, and the micropolitics of grassroots organizing in Mexico.

As states weaken and public health care deteriorates throughout the developing world, new spaces are opening for civil society groups to fill the gaps of declining health systems. In Mexico, popular health groups have responded to health care decline by building community clinics, establishing health promoter training programs, and opening natural medicine pharmacies. Lower- and working-class women are the primary participants in these groups that use a self-help approach to find practical solutions to local health care problems. However, little is known about participants' circumstances, motivations, or the ideals they embrace. Drawing from women's narratives, I explore the "micropolitics" of women's participation in local health groups. I examine their efforts to reclaim control over the health process as a metaphor for claiming control over their lives. Highlighting the "instrumental effects" of participants' medical encounters and healing relationships, this case illuminates some unintended consequences of health care decline.

A pulsed-dose study evaluating chronic toxicity of chlorothalonil to fish: A case study for environmental risk assessment.

Chlorothalonil is a fungicide which is highly toxic to aquatic organisms. However, in natural aquatic environments, it is very rapidly degraded, with a half-life typically in hours, reducing exposure of aquatic organisms and the potential for effects. In standard regulatory studies looking at the chronic toxicity of chlorothalonil to fathead minnow, the most sensitive endpoint was fecundity. A standard fish full-life cycle study, where chlorothalonil concentrations were maintained constant throughout, resulted in a no-observed-effect concentration (NOEC) of 1.4 µg/L. Comparing peak modeled exposure concentrations to this NOEC can result in the chronic risk to fish being considered unacceptable. The present study investigated the effect on fecundity in fathead minnow using a fish short-term reproduction assay. Five different exposure profiles were employed with time-varying concentrations based on realistic worst-case modeled environmental exposure profiles, multiplied by an assessment factor of 10, which resulted in maximum measured concentrations up to 15.5 µg/L. There were no effects on fecundity from any of the exposure profiles tested. Therefore, based on these more realistic exposure profiles, the chronic risk to fish could be considered acceptable if these exposures were deemed to be representative of the worst case. Environ Toxicol Chem 2019;38:1549-1559. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Development of an in vitro diagnostic method to determine the genotypic sex of Xenopus laevis.

A genotypic sex determination assay provides accurate gender information of individuals with well-developed phenotypic characters as well as those with poorly developed or absent of phenotypic characters. Determination of genetic sex for Xenopus laevis can be used to validate the outcomes of Tier 2 amphibian assays, and is a requirement for conducting the larval amphibian growth and development assay (LAGDA), in the endocrine disruptor screening program (EDSP), test guidelines. The assay we developed uses a dual-labeled TaqMan probe-based real-time polymerase chain reaction (real-time PCR) method to determine the genotypic sex. The reliability of the assay was tested on 37 adult specimens of X. laevis collected from in-house cultures in Eurofins EAG Agroscience, Easton. The newly designed X. laevis-specific primer pair and probe targets the DM domain gene linked-chromosome W as a master female-determining gene. Accuracy of the molecular method was assessed by comparing with phenotypic sex, determined by necropsy and histological examination of gonads for all examined specimens. Genotypic sex assignments were strongly concordant with observed phenotypic sex, confirming that the 19 specimens were male and 18 were female. The results indicate that the TaqMan® assay could be practically used to determine the genetic sex of animals with poorly developed or no phenotypic sex characteristics with 100% precision. Therefore, the TaqMan® assay is confirmed as an efficient and feasible method, providing a diagnostic molecular sex determination approach to be used in the amphibian endocrine disrupting screening programs conducted by regulatory industries. The strength of an EDSP is dependent on a reliable method to determine genetic sex in order to identify reversals of phenotypic sex in animals exposed to endocrine active compounds.