Antinociceptive Analysis of Natural Monoterpenes Eugenol, Menthol, Carvacrol and Thymol in a Zebrafish Larval Model.

In the last decade, a considerable number of studies have broadened our knowledge of the nociceptive mechanisms of pain, a global health problem in both humans and animals. The use of herbal compounds such as eugenol, menthol, thymol, and carvacrol as analgesic agents has accompanied the growing interest in this area, offering a possible solution for this complex problem. Here, we aimed to explore how these natural substances-at three different concentrations (2, 5 and 10 mg/L)-affect the pain responses in zebrafish (Danio rerio) larvae exposed to 0.05% acetic acid (AA) for 1 min. By analysing the activity of acetylcholinesterase (AChE), 5'-ectonucleotidase and NTPDases, as well as aversion and exploratory behaviours, it was observed that that although all substances were effective in counteracting the pain stimulus, the concentration range within which they do so might be very limited. Eugenol, despite its acknowledged properties in fish anaesthesia, failed to alleviate the pain stimulus at low concentrations. Contrastingly, menthol exhibited the most promising results at the lowest concentrations tested. Overall, it is concluded that menthol might be a good analgesic for this species, qualifying it as a substance of interest for prospective studies.

Non-Invasive Biomarkers in Saliva and Eye Infrared Thermography to Assess the Stress Response of Calves during Transport.

Animal transport is currently a stressful procedure. Therefore, animal-based indicators are needed for reliable and non-invasive welfare assessment. Saliva is a biospecimen with potential validity for the determination of cortisol and oxidative stress, although its use to assess calf welfare during transport has never been tested. Similarly, the applicability and reliability of infrared thermography to assess temperature change during calves' transport have never been evaluated. These objectives were outlined following the known and growing need to identify non-invasive methodologies for stress assessment in bovines. This study was conducted on 20 calves of the Arouquesa autochthone breed, at about nine months of age, during their transport to slaughter. For each animal, saliva samples and thermographic images of the eye were collected at three time points: before transport, after transport, and at slaughter. The saliva was then processed to measure cortisol levels and oxidative stress parameters (reactive oxygen species, thiobarbituric acid reactive substance, carbonyls, and advanced oxidation protein products), and the images were analyzed using FLIR Tools+ software. There was an increase in cortisol concentration and oxidative stress parameters (reactive oxygen species, thiobarbituric acid reactive substance, carbonyls, and advanced oxidation protein products) in saliva after transport. An increase in eye temperature triggered by transport was also observed. The cortisol and eye temperature results at slaughter were returned to values similar to those before transport; however, the values of oxidative stress remained increased (mainly TBARS values). These non-invasive techniques seem to be reliable indicators of stress in bovine transport, and oxidative stress parameters in saliva may be a persistent marker for welfare assessment.

Beneficial Effects of Broccoli (Brassica oleracea var italica) By-products in Diet-induced Obese Mice.

BACKGROUND/AIM: Obesity currently affects the whole world, with greater incidence in high-income countries, with vast economic and social costs. Broccoli harvest generates many by-products equally rich in bioactive compounds with potential anti-obesity effects. This study aimed to evaluate the anti-obesity effects of broccoli by-products flour (BF) in obese mice. MATERIALS AND METHODS: A commercial high-fat diet formulation (representing a Western diet) was used to induce obesity in mice. BF (0.67% or 1.34% weight/weight) was incorporated as a chemoprevention compound into a control and a hypercholesterolemic diet, at two different concentrations, and fed for 14 weeks to C57BL/6J mice. For a therapeutic approach, two groups were fed with the hypercholesterolemic diet for 10 weeks, and then fed with BF-supplemented diets in the last 4 weeks of the study. RESULTS: BF supplementation helped to maintain a lower body weight, reduced adipose tissue accumulation, and enhanced the basal activity of superoxide dismutase and glutathione S-transferase. Although BF supplementation tended to reduce the relative liver weight increased by the Western diet, the differences were not significant. CONCLUSION: BF appears to have a beneficial effect in preventing weight gain and fat accumulation induced by hypercholesterolemic diets.

Enantioselective Ecotoxicity of Venlafaxine in Aquatic Organisms: Daphnia and Zebrafish.

Venlafaxine is a chiral antidepressant detected in aquatic compartments. It was recently included in the 3rd Watch List from the European Union. The present study aimed to investigate venlafaxine toxicity effects, targeting possible enantioselective effects, using two aquatic organisms, daphnia (Daphnia magna) and zebrafish (Danio rerio). Specimens were exposed to both racemate, (R,S)-venlafaxine (VEN), and to pure enantiomers. Acute assays with daphnia showed that up to 50 000 µg/L of the (R,S)-VEN induced no toxicity. Organisms were also exposed to sublethal concentrations (25-400 µg/L) of (R,S)-, (R)- and (S)-VEN, for 21 days. No significant effects on mortality, age at first reproduction, and size of the first clutch were observed. However, a decrease in fecundity was observed for both enantiomers at the highest concentration. Regarding zebrafish, the effects of venlafaxine on mortality, embryo development, behavior, biochemistry, and melanin pigmentation were investigated after 96 h of exposure to the range of 0.3-3000 µg/L. (R)-VEN significantly increased the percentage of malformations in comparison with (S)-VEN. Behavior was also enantiomer dependent, with a decrease in the total distance moved and an increase in avoidance behavior observed in organisms exposed to (R)-VEN. Despite the biochemical variations, no changes in redox homeostasis were observed. (R)-VEN also led to an increase in zebrafish pigmentation. The different susceptibility to venlafaxine and enantioselective effects were observed in zebrafish. Our results suggest that at environmental levels (R,S)-VEN and pure enantiomers are not expected to induce harmful effects in both organisms, but (R)-VEN increased malformations in zebrafish larvae, even at reported environmental levels. These results highlight the importance of including enantioselective studies for an accurate risk assessment of chiral pollutants. Environ Toxicol Chem 2022;41:1851-1864. © 2022 SETAC.

Neuroendocrine and Eosinophilic Granule Cells in the Gills of Tilapia, Oreochromis niloticus: Effects of Waterborne Copper Exposure.

The contamination of aquatic ecosystems with copper (Cu) poses a serious threat to aquatic organisms. Although the histopathological changes caused by Cu in fish gills are well documented, knowledge about the impact of this metal in gill specific cell types, such as neuroendocrine cells (NECs) and eosinophilic granule cells (EGCs), is still limited. In the present work, Nile tilapia (Oreochromis niloticus) were exposed for 21 days to nominal concentrations of Cu (40 and 400 µg L(-1)). Stereological methods were used to estimate the volumetric density of both NECs and EGCs in fish gill filament after 3, 7, 14, and 21 days of exposure. The results showed that Cu significantly increased the relative volume of NECs, whereas the relative volume of EGCs decreased. NECs were more affected by Cu in the first 7 days of exposure, during which a greater increase in their relative volume was observed. The Cu exposure induced a progressive decrease in the relative volume of EGCs, which reached statistical significance after 14 days of exposure. An exception was observed in subepithelial EGCs with a slight increase in their relative volume after 3 days of exposure. Our findings confirm that Cu can modulate both neuroendocrine and immune systems and becomes immunotoxic after a prolonged exposure.

An immunohistochemical study of gill epithelium cells in the Nile tilapia, Oreochromis niloticus.

This study reports the first complete mapping of the gill epithelium in a tilapia species. Different gill epithelial cell types of the Nile tilapia, Oreochromis niloticus L. have been identified and located using different antisera against mammalian proteins and various histochemical techniques: Periodic Acid Schiff (PAS), Alcian Blue pH 1.0, 2.5, 3.5, Giemsa and Grimelius. The results show that the stratified filament epithelium of O. niloticus gill can be divided into two distinct regions, a superficial layer, where pavement, mucous and mitochondria rich cells can be found, and a deep layer, constituted by undifferentiated, myoepithelial-like, granular and neuroendocrine cells. V-ATPase and Na+/K+-ATPase presence allowed the identification of pavement and mitochondria-rich cells, respectively, suggesting that, in O. niloticus, pavement cells are implicated in Na+ uptake, whereas mitochondria-rich cells have a role in Cl- uptake. The use of PAS and Alcian Blue allowed the recognition of different sub-populations of mucous cells that differentiate from a common deeper precursor. Neuroendocrine markers were detected in different cell populations, stating evidence for a neuroendocrine role of mitochondria- rich cells, and suggesting the existence of distinct neural pathways, a putative O2-chemosensory and an ion regulatory pathway. A defence role was attributed to the deep filament epithelium, suggested by the presence of resident giemsa positive- eosinophil granular cells. The antibody raised against proliferating cell nuclear antigen identified two different cell types, the undifferentiated cells and myoepithelial-like cells. In the superficial layer, it is here stated for the first time the existence of vimentin positive support cells.

Fine structure of the branchial epithelium in the teleost Oreochromis niloticus.

We have studied the gill epithelium of Oreochromis niloticus using transmission electron microscopy with the particular interested relationship between cell morphology and osmotic, immunoregulatory, or other non-regulatory functions of the gill. Pavement cells covered the filament epithelium and lamellae of gills, with filament pavement cells showing distinct features from lamellar pavement cells. The superficial layer of the filament epithelium was formed by osmoregulatory elements, the columnar mitochondria-rich, mucous and support cells, as well as by their precursors. Light mitochondria-rich cells were located next to lamellae. They exhibited an apical crypt with microvilli and horizontal small dense rod-like vesicles, sealed by tight junctions to pavement cells. Dark mitochondria-rich cells had long dense rod-like vesicles and a small apical opening sealed by tight junctions to pavement cells. The deep layer of the filament epithelium was formed by a network of undifferentiated cells, containing neuroepithelial and myoepithelial cells, macrophage and eosinophil-like cells and their precursors, as well as precursors of mucous cells. The lateral-basal surface was coated by myoepithelial cells and a basal lamina. The lamellar blood lacunae was lined by pillar cells and surrounded by a basal lamina and pericytes. The data presented here support the existence of two distinct types of pavement cells, mitochondria-rich cells, and mitochondria-rich cells precursors, a structural role for support cells, a common origin for pavement cells and support cells, a paracrine function for neuroepithelial cells in the superficial layer, and the control of the lamellar capillary base by endocrine and contractile cells. Data further suggest that the filament superficial layer is involved in gill osmoregulation, that may interact, through pale mitochondria-rich cells, with the deep layer and lamellae, whereas the deep layer, through immune and neuroendocrine systems, acts in the regeneration and defense of the tissue.

Copper toxicity in gills of the teleost fish, Oreochromis niloticus: effects in apoptosis induction and cell proliferation.

Recent in vitro studies have demonstrated that copper may induce apoptosis triggering the activation of caspase-3, a central effector of apoptotic cell death. However, the precise mechanism of copper-induced apoptosis is still unclear, even less so in Oreochromis niloticus where no caspase genes have been reported so far. This study aimed to assess the in vivo role of copper in apoptosis induction on O. niloticus gill, simultaneously contributing to elucidate the mechanism of copper-induced apoptosis. Caspase-3 gene was partially sequenced and, after in vivo exposures to 40 and 400 microgL(-1) of copper, its mRNA expression was evaluated by real-time PCR. Apoptosis was also evaluated by TUNEL assay and cell proliferation identified using an antibody against proliferating cell nuclear antigen (PCNA). The copper concentrations used did not induce the upregulation of caspase-3 gene in O. niloticus gill. In addition, in the gills of fish exposed to copper there was no increase in the estimated relative volume of apoptotic cells, indicating that neither the caspase-3-dependent or caspase-independent apoptotic pathways were induced. On the other hand, the increase in the volumetric density of epithelial proliferating cells suggests a concentration-dependent repair response.

Histopathological gill changes in wild leaping grey mullet (Liza saliens) from the Esmoriz-Paramos coastal lagoon, Portugal.

The histopathological changes are among the most recognized responses to environmental stressors, namely heavy metals. Liza saliens were sampled, in the Portuguese Esmoriz-Paramos coastal lagoon to assess their gill histopathological response to Cu and Zn contaminated sediments. A lesion prevalence index, severity, and extension scores of each lesion, as well as an assessment value (severity x extension), were determined to evaluate the effect of environmental heavy metal exposure. The main histopathological changes observed were aneurisms, hyperplasia, lifting, and vasodilation. A high prevalence for each lesion (65-85%) was found, in addition to a high number of simultaneous lesions. Vasodilation and hyperplasia were the lesions that showed higher prevalence indexes. Lifting, followed by hyperplasia, were the lesions with highest assessment value. The prevalence of the number of the lesions found in L. saliens collected from the Esmoriz-Paramos lagoon was high when compared with mullet caught in the sea. A previous work revealed that fish collected in the lagoon showed elevated Cu and Zn levels in their gills, in a positive correlation with age. However, no positive correlation was observed between assessment value and gill metal levels.