Development of a thin-film solid-phase microextraction (TF-SPME) method coupled to liquid chromatography and tandem mass spectrometry for high-throughput determination of steroid hormones in white sucker fish plasma.

Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17ß-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150 ng/mL and from 0.020 to 0.500 ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract.

Maternal Transfer and Effects of Selenium on Early Life Stage Development of Redside Shiner (Richardsonius balteatus).

Maternal transfer of selenium (Se) to developing fish eggs during vitellogenesis can cause larval deformity and mortality. Previous studies have shown wide variation among fish species in both the magnitude of maternal transfer (exposure) and the egg Se concentration causing effects (sensitivity). We studied maternal transfer and effects of Se on early life stage development, survival, and growth of redside shiner (Richardsonius balteatus), a small-bodied cyprinid that has been reported to have relatively high ovary:muscle Se concentration ratios. Gametes were collected from lentic areas in southeast British Columbia (Canada) with a range of dietary Se concentrations related to weathering of waste rock from coal mining. Eggs were fertilized and reared in the laboratory from hatch to the onset of exogenous feeding. Larvae were assessed for survival, length, weight, Se-characteristic deformities, and edema. Eggs from a total of 56 females were collected, with egg Se concentrations from 0.7 to 28 mg/kg dry weight. Maternal transfer varied among sites, with egg:muscle Se concentration ratios ranging from <1 to >4. We also found that sampling residual ovaries can overestimate Se concentrations in ripe eggs by up to a factor of 5.7. A correlation between larval weight and egg Se concentration was identified, although the relationship was weak (r2 < 0.1) and appeared to be a site effect. No other relationships were observed between larval endpoints and egg Se concentrations up to the highest concentration tested, indicating that the effects threshold for this species may be >28 mg/kg dry weight in eggs. These data indicate that redside shiner is less sensitive to maternally transferred Se than most other tested fish species. Environ Toxicol Chem 2023;42:2350-2357. © 2023 SETAC.

Reactive oxygen species and nitric oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of papaver.

Pollen-pistil interactions are critical early events regulating pollination and fertilization. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants. Although data implicate the involvement of reactive oxygen species (ROS) and nitric oxide (NO) in pollen-pistil interactions and the regulation of pollen tube growth, there has been a lack of studies investigating ROS and NO signaling in pollen tubes in response to defined, physiologically relevant stimuli. We have used live-cell imaging to visualize ROS and NO in growing Papaver rhoeas pollen tubes using chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and demonstrate that SI induces relatively rapid and transient increases in ROS and NO, with each showing a distinctive "signature" within incompatible pollen tubes. Investigating how these signals integrate with the SI responses, we show that Ca(2+) increases are upstream of ROS and NO. As ROS/NO scavengers alleviated both the formation of SI-induced actin punctate foci and also the activation of a DEVDase/caspase-3-like activity, this demonstrates that ROS and NO act upstream of these key SI markers and suggests that they signal to these SI events. These data represent, to our knowledge, the first steps in understanding ROS/NO signaling triggered by this receptor-ligand interaction in pollen tubes.

Hepatic transcriptomics and protein expression in rainbow trout exposed to municipal wastewater effluent.

Municipal wastewater effluents (MWWEs) represent one of the largest point sources of contamination, but few studies have addressed the impact on fish populations. We tested the hypothesis that MWWEs disrupt multiple stress-related pathways by examining expression of genes and proteins in rainbow trout (Oncorhynchus mykiss). A caging study was undertaken by placing juvenile trout for 14 d either at an upstream control or 100%, 50%, and 10% MWWE sites. A custom-made low-density rainbow trout cDNA microarray was utilized for transcriptomics, and select gene expression was confirmed with quantitative real-time PCR. MWWE exposure significantly elevated plasma cortisol, glucose, and vitellogenin levels, and altered the expression of a number of hepatic genes. Notably, expression of stress-related genes, hormone receptors, glucose transporter 2, and genes related to immune function were altered. The gene and protein expression of glucocorticoid receptor, heat shock proteins 70 and 90, and cytochrome P4501A1 were also impacted by MWWE exposure. Our results demonstrate that tertiary-treated MWWEs elicit an organismal and cellular stress response in trout and may lead to an enhanced energy demand in the exposed fish. The disruption in multiple stress-related pathways suggests that tertiary-treated MWWEs exposure may reduce fish performance to subsequent stressors.

In vivo solid-phase microextraction sampling combined with metabolomics and toxicological studies for the non-lethal monitoring of the exposome in fish tissue.

Various environmental studies have employed the biomonitoring of fish in their aquatic ecosystems in order to identify potential metabolic responses to the exposome. In this study, we applied in vivo solid-phase microextraction (SPME) to perform non-lethal sampling on the muscle tissue of living fish to extract toxicants and various endogenous metabolites. Sixty white suckers (Catastomus commersonii) were sampled from sites upstream, adjacent, and downstream from the oil sands development region of the Athabasca River (Alberta, Canada) in order to track their biochemical responses to potential contaminants. In vivo SPME sampling facilitated the extraction of a wide range of endogenous metabolites, mainly related to lipid metabolism. The obtained results revealed significant changes in the levels of numerous metabolites, including eicosanoids, linoleic acids, and fat-soluble vitamins, in fish sampled in different areas of the river, thus demonstrating SPME's applicability for the direct monitoring of exposure to different environmental toxicants. In addition, several classes of toxins, including petroleum-related compounds, that can cause serious physiological impairment were tentatively identified in the extracts. In vivo SPME, combined with the analysis of contaminants and endogenous metabolites, provided important information about the exposome; as such, this approach represents a potentially powerful and non-lethal tool for identifying the mechanisms that produce altered metabolic pathways in response to the mixtures of different environmental pollutants.

In vivo microsampling to capture the elusive exposome.

Loss and/or degradation of small molecules during sampling, sample transportation and storage can adversely impact biological interpretation of metabolomics data. In this study, we performed in vivo sampling using solid-phase microextraction (SPME) in combination with non-targeted liquid chromatography and high-resolution tandem mass spectrometry (LC-MS/MS) to capture the fish tissue exposome using molecular networking analysis, and the results were contrasted with molecular differences obtained with ex vivo SPME sampling. Based on 494 MS/MS spectra comparisons, we demonstrated that in vivo SPME sampling provided better extraction and stabilization of highly reactive molecules, such as 1-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoleoyl-glycero-3-phosphocholine, from fish tissue samples. This sampling approach, that minimizes sample handling and preparation, offers the opportunity to perform longitudinal monitoring of the exposome in biological systems and improve the reliability of exposure-measurement in exposome-wide association studies.

Physiological and growth responses to water deficit in the bioenergy crop Miscanthus x giganteus.

High yielding perennial biomass crops of the species Miscanthus are widely recognized as one of the most promising lignocellulosic feedstocks for the production of bioenergy and bioproducts. Miscanthus is a C4 grass and thus has relatively high water use efficiency. Cultivated Miscanthus comprises primarily of a single clone, Miscanthus x giganteus, a sterile hybrid between M. sacchariflorus and M. sinensis. M. x giganteus is high yielding and expresses desirable combinations of many traits present in the two parental species types; however, it responds poorly to low water availability. To identify the physiological basis of the response to water stress in M. x giganteus and to identify potential targets for breeding improvements we characterized the physiological responses to water-deficit stress in a pot experiment. The experiment has provided valuable insights into the temporal aspects of drought-induced responses of M. x giganteus. Withholding water resulted in marked changes in plant physiology with growth-associated traits among the first affected, the most rapid response being a decline in the rate of stem elongation. A reduction in photosynthetic performance was among the second set of changes observed; indicated by a decrease in stomatal conductance followed by decreases in chlorophyll fluorescence and chlorophyll content. Measures reflecting the plant water status were among the last affected by the drought treatment. Metabolite analysis indicated that proline was a drought stress marker in M. x giganteus, metabolites in the proline synthesis pathway were more abundant when stomatal conductance decreased and dry weight accumulation ceased. The outcomes of this study in terms of drought-induced physiological changes, accompanied by a proof-of-concept metabolomics investigation, provide a platform for identifying targets for improved drought-tolerance of the Miscanthus bioenergy crop.

Tissue-specific metabolic changes in response to an acute handling disturbance in juvenile rainbow trout exposed to municipal wastewater effluent.

The objective of this study was to evaluate the effects of municipal wastewater effluent (MWWE) exposure on aspects of both organismal and cellular stress response in rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were exposed for 14 d (2-d static renewal) to tertiary-treated MWWE at concentrations of 0%, 20% and 90%. Following the MWWE exposure, fish were subjected to an acute handling stress and sampled at 1, 4 and 24 h post-stressor, to evaluate the fish performance to additional stressors. Organismal stress response evaluation included measuring plasma cortisol, glucose and lactate concentrations, and tissue metabolic capacity, including gluconeogenic (liver) and glycolytic enzyme activities in the liver, brain, heart and gill. No significant differences between treatments were seen in plasma cortisol, glucose or lactate concentrations after 14 d exposure to MWWE. However, MWWE exposure significantly affected plasma cortisol and glucose response to the acute secondary stressor. Acute handling disturbance enhanced liver gluconeogenic capacity in the control group, but this response was altered in the MWWE exposed groups. MWWE exposure did not affect the acute stressor-mediated enhancement of brain or gill glycolytic capacity, but significantly reduced the glycolytic capacity of liver and heart in response to a secondary stressor compared to the control group. Altogether, chronic exposure to MWWE impacts the metabolic performances to a secondary stressor challenge and this includes disruptions in tissue-specific gluconeogenic and glycolytic capacities in rainbow trout.

Temporal changes in stress and tissue-specific metabolic responses to municipal wastewater effluent exposure in rainbow trout.

Sub-chronic exposure to municipal wastewater effluent (MWWE) in situ was recently shown to impact the acute response to a secondary stressor in rainbow trout (Oncorhynchus mykiss). However, little is known about whether MWWE exposure in itself is stressful to the animal. To address this, we carried out a laboratory study to examine the organismal and cellular stress responses and tissue-specific metabolic capacity in trout exposed to MWWE. Juvenile rainbow trout were exposed to 0, 20 and 90% MWWE (from a tertiary wastewater treatment plant), that was replenished every 2d, for 14 d. Fish were sampled 2, 8 or 14 d post-exposure. Plasma cortisol, glucose and lactate levels were measured as indicators of organismal stress response, while inducible heat shock protein 70 (hsp70), constitutive heat shock protein 70 (hsc70) and hsp90 expression in the liver were used as markers of cellular stress response. Impact of MWWE on cortisol signaling was ascertained by determining glucocorticoid receptor protein (GR) expression in the liver, brain and, heart, and metabolic capacity was evaluated by measuring liver glycogen content and tissue-specific activities of key enzymes in intermediary metabolism. Plasma glucose and lactate levels were unaffected by exposure to MWWEs, whereas cortisol showed a transient increase in the 20% group at 8d. Liver hsc70 and hsp90, but not hsp70 expression, were higher in the 90% MWWE group after 8d. There was a temporal change in GR expression in the liver and heart, but not brain of trout exposed to MWWE. Liver glycogen content and activities liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), and alanine aminotransferase (AlaAT) were significantly affected by MWWE exposure. The glycolytic enzymes pyruvate kinase (PK) and hexokinase (HK) activities were significantly higher temporally by MWWE exposure in the gill and heart, but not in the liver and brain. Overall, a 14 d exposure to MWWE evokes a cellular stress response and perturbs the cortisol stress response in rainbow trout. The tissue-specific temporal changes in the metabolic capacity suggest enhanced energy demand in fish exposed to MWWE, which may eventually lead to reduced fitness.

Exposure to municipal wastewater effluent impacts stress performance in rainbow trout.

The objective of the study was to examine the impact of municipal wastewater effluents on the functioning of the cortisol stress axis in rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were caged upstream (reference) and downstream (100% and 10% effluent) of a tertiary-treated municipal wastewater treatment plant outfall and sampled at 14d later (0 time samples). A second set of fish were then subjected to a 5 min handling disturbance and sampled at 1 and 24h post-stressor exposure. Plasma cortisol, glucose and lactate concentrations, liver and brain glucocorticoid receptor (GR) protein levels, head kidney mRNA abundances of corticosteroidogenesis genes, including steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), 11ß-hydroxylase and melanocortin 2 receptor (MC2R), and key liver metabolic enzyme activities, were measured. Exposure to effluent for 14d significantly elevated plasma cortisol and lactate levels in 100% effluent group compared to the reference and 10% effluent sites. There was a significantly higher StAR mRNA abundance in the effluent groups compared to the upstream control. GR protein levels in the liver, but not the brain, were significantly higher in the 100% effluent group compared to the upstream control group. Chronic exposure to 100% effluent for 14d significantly lowered liver hexokinase and glucokinase activities, but did not affect glycogen content or the activities of phosphoenolpyruvate carboxykinase, pyruvate kinase, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase compared to the other two groups. Subjecting these fish to a secondary acute stressor elicited a physiological stress response, including significant transient elevation in plasma cortisol, glucose and lactate levels at 1h which dropped to pre-stress levels at 24h after stressor exposure, in the control and 10% effluent groups, but this conserved stress response was impaired in the 100% effluent group. The 100% effluent group fish also had significantly higher StAR and P450scc mRNA abundances at 1h post-stress, while transcript abundances of all the major corticosteroidogenesis genes were suppressed at 24h post-stressor compared to the control and 10% effluent groups. Considered together, exposure to full-strength MWWE for 14d elicits a chronic stress response in rainbow trout, and perturbs the conserved adaptive response to an acute stressor. Our results reveal that the impact of tertiary-treated MWWE on stress performance in rainbow trout is abolished by 90% effluent dilution.

Biomonitoring of perfluorochemicals and toxicity to the downstream fish community of Etobicoke Creek following deployment of aqueous film-forming foam.

On August 2, 2005, Air France Flight 358 descended on Lester B. Pearson International Airport (Toronto, ON, Canada) during adverse weather conditions and overran the runway, leading to an onboard fire which destroyed the aircraft. Large quantities (48000L) of aqueous film-forming foam (AFFF) were applied to the burning fuselage within meters of Etobicoke Creek. Local authorities could not confirm the identity of the AFFF formulation applied, but chemical analyses of fish livers collected 9 days post-AFFF application indicated that no perfluorinated acids (PFAs) were elevated at the site of application or downstream. This, and higher concentrations of a fluorotelomer sulfonate in fish liver collected downstream, suggests that an AFFF containing telomerized polyfluorinated material was likely used. However, as an urbanized stream within a heavily developed commercial, industrial, and residential watershed, background levels of perfluorinated compounds in Etobicoke Creek were considerable at all sites. Enlarged fish livers adjacent the AFFF-application site, commensurate with depressed peroxisomal beta-oxidation and hepatic oxidative stress, demonstrate some short-term impact of the AFFF on exposed fish within 9 days of its release. Most fish biochemical responses had recovered to normal values by 120 days, although there was some indication that AFFF-associated contamination shifted further downstream over this interval. This study suggests contemporary AFFFs exert relatively low toxicity on fish communities under realistic exposure scenarios.

Adrenocorticotropic hormone suppresses gonadotropin-stimulated estradiol release from zebrafish ovarian follicles.

While stress is known to impact reproductive performance, the pathways involved are not entirely understood. Corticosteroid effects on the functioning of the hypothalamus-pituitary-gonadal axis are thought to be a key aspect of stress-mediated reproductive dysfunction. A vital component of the stress response is the pituitary secretion of adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. We recently reported MC2R mRNA abundance in fish gonads leading to the hypothesis that ACTH may be directly involved in gonadal steroid modulation. Using zebrafish (Danio rerio) ovarian follicles, we tested the hypothesis that acute ACTH stimulation modulates cortisol and estradiol (E(2)) secretion. ACTH neither affected cortisol nor unstimulated E(2) release from ovarian follicles. However, ACTH suppressed human chorionic gonadotropin (hCG)-stimulated E(2) secretion in a dose-related manner, with a maximum decrease of 62% observed at 1 I.U. ACTH mL(-1). This effect of ACTH on E(2) release was not observed in the presence of either 8-bromo-cAMP or forskolin, suggesting that the mechanism(s) involved in steroid attenuation was upstream of adenylyl cyclase activation. Overall, our results suggest that a stress-induced rise in plasma ACTH levels may initiate a rapid down-regulation of acute stimulated E(2) biosynthesis in the zebrafish ovary, underscoring a novel physiological role for this pituitary peptide in modulating reproductive activity.

Characterization of the mRNA expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles.

The objective of this study was to investigate the levels of expression of steroid biosynthetic enzymes and steroidogenic acute regulatory protein (StAR) at different stages of ovarian follicular development in zebrafish (Danio rerio), and to investigate the sites within the steroid biosynthetic pathway that may be regulated by gonadotropins. Ovarian follicles of sexually mature fish were separated into primary, previtellogenic, vitellogenic, and mature stages and the expression of StAR, P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 hydroxylase/lyase (P450c17), 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), and P450 aromatase (P450aromA) was determined by Real time RT-PCR. The expression of all genes changed significantly as follicles grew, with a decrease in the expression of StAR, P450scc, 3beta-HSD and P450c17 with maturation, and an increase in the expression of 17beta-HSD3 during vitellogenesis and 17beta-HSD1 and P450aromA during previtellogenesis. In vitro incubation of vitellogenic follicles demonstrated that the expression of StAR, 17beta-HSD3, and P450aromA increased in response to hCG, and decreased in the absence of hCG. In contrast, the expression of P450scc, 3beta-HSD, P450c17, and 17beta-HSD1 remained constant between treatments and over time. Testosterone and estradiol production in the culture medium was stimulated by human chorionic gonadotropin (hCG). These experiments aid in the characterization of the roles and regulation of steroids throughout ovarian development, and suggest that gonadotropins play a key role in the regulation of StAR, 17beta-HSD3, and P450aromA in zebrafish.