Behavioral Effects of the Mixture and the Single Compounds Carbendazim, Fipronil, and Sulfentrazone on Zebrafish (Danio rerio) Larvae.

Pesticides are often detected in freshwater, but their impact on the aquatic environment is commonly studied based on single compounds, underestimating the potential additive effects of these mixtures. Even at low concentrations, pesticides can negatively affect organisms, altering important behaviors that can have repercussions at the population level. This study used a multi-behavioral approach to evaluate the effects of zebrafish larvae exposure to carbendazim (C), fipronil (F), and sulfentrazone (S), individually and mixed. Five behavioral tests, thigmotaxis, touch sensitivity, optomotor response, bouncing ball test, and larval exploratory behavior, were performed to assess potential effects on anxiety, fear, and spatial and social interaction. Significant changes were observed in the performance of larvae exposed to all compounds and their mixtures. Among the single pesticides, exposure to S produced the most behavioral alterations, followed by F and C, respectively. A synergistic effect between the compounds was observed in the C + F group, which showed more behavioral effects than the groups exposed to pesticides individually. The use of behavioral tests to evaluate pesticide mixtures is important to standardize methods and associate behavioral changes with ecologically relevant events, thus creating a more realistic scenario for investigating the potential environmental impacts of these compounds.

Assessment of toxicity of pyriproxyfen, Bacillus thuringiensis, and malathion and their mixtures used for mosquito control on embryo-larval development and behavior of zebrafish.

Pyriproxyfen (PPF), Bacillus thuringiensis israelensis (BTI), and malathion (MLT) are widely used worldwide to control the population of mosquitos that transmit arboviruses. The current work aimed to evaluate the toxicity of these single pesticides and their binary mixtures of PPF + BTI, PPF + MLT, and MLT + BTI on the embryo-larval stage of zebrafish (Danio rerio) as an animal model. Epiboly, mortality, apical endpoints, affected animals, heart rate, morphometric, thigmotaxis, touch sensitivity, and optomotor response tests were evaluated. PPF and MLT and all mixtures reduced the epiboly percentage. Mortality increased significantly in all exposed groups, except BTI, with MLT being the most toxic. The observed apical endpoints were pericardial and yolk sac edemas, and tail and spine deformation. Exposure to MLT showed a higher percentage of affected animals. A reduction in heart rate was also observed in MLT- and PPF + MLT-exposed groups. The PPF + MLT mixture decreased head measurements. Behavioral alterations were observed, with a decrease in thigmotaxis and touch sensitivity responses in PPF + MLT and MLT + BTI groups. Finally, optomotor responses were affected in all groups. The above data obtained suggest that the MLT + PFF mixture has the greatest toxicity effects. This mixture affected embryo-larval development and behavior and is close to the reality in several cities that use both pesticides for mosquito control rather than single pesticides, leading to a reevaluation of the strategy for mosquito control.

Essential Oils Produce Developmental Toxicity in Zebrafish Embryos and Cause Behavior Changes in Zebrafish Larvae.

Essential oils have gained significant popularity in various industries due to their biological properties, but their potential toxic effects on living organisms have been poorly investigated. This study aimed to evaluate the effects of lemongrass, thyme, and oregano essential oils on zebrafish embryos and larvae as animal models. Embryos were exposed to different concentrations of essential oils, and various endpoints were assessed, including epiboly, mortality (LC50), morphometry, and behavioral changes. All three essential oils reduced epiboly, affecting embryonic development. LC50 values were calculated for lemongrass (3.7 µg/mL), thyme (14.4 µg/mL), and oregano (5.3 µg/mL) oils. Larvae exposed to these oils displayed morphological defects, including growth reduction, spinal deformation, pericardial edema, eye size reduction, and reduced swim-bladder inflation. Morphometric analysis confirmed reduced larval length at higher oil concentrations. Essential-oil exposure altered zebrafish larval swimming behavior, with lemongrass oil reducing dark-cycle activity and oregano oil increasing light-cycle activity, suggesting neurodevelopmental toxicity. These findings illustrate the adverse effects of these oils on zebrafish embryos and larvae and reveal essential-oil toxicity, indicating careful use should be considered, particularly during pregnancy.

Efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates.

The aim of this study was to evaluate the efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates. Initially, 24-1 fractional experimental design was carried out to obtain an optimized formulation of liposomes containing CPO (CPO-LipoC), which were then used to prepare stealth liposomes (CPO-LipoS). Liposomal formulations were characterized by their mean size diameter, polydispersity index (PDI), and drug encapsulation efficiency (EE%). Immunosuppressed mice were exposed to CPO-LipoS at 0.5 mg/kg/day for 14 days to verify possible histopathological alterations in the liver and kidneys. Immunosuppressed mice infected with C. neoformans were treated with CPO-LipoS at 0.5 mg/kg/day for 14 days to quantify the fungal burden in spleen, liver, lungs, and brain. CPO-LipoS presented a mean size diameter, PDI, and EE% of 101.4 ± 0.7 nm, 0.307, and 96.4 ± 0.9%, respectively. CPO-LipoS was non-toxic for the liver and kidneys of immunosuppressed mice. At the survival curve, all infected animals submitted to treatment with CPO-LipoS survived until the end of the experiment. Treatment with CPO-LipoS reduced C. neoformans cells in the spleen (59.3 ± 3.4%), liver (75.0 ± 3.6%), lungs (75.7 ± 6.7%), and brain (54.2 ± 3.2%). CPO-LipoS exhibit antifungal activity against C. neoformans, and the encapsulation of CPO into stealth liposomes allows its use as a systemic drug for treating cryptococcosis.

Single and joint toxic effects of thyroid hormone, levothyroxine, and amiodarone on embryo-larval stages of zebrafish (Danio rerio).

This study evaluates single and joint endocrine disruptor toxicities of thyroid hormone, levothyroxine, and amiodarone in the embryo-larval stages of Danio rerio. Single toxicity experiments were carried out in concentrations based on the environmental concentration and increasing concentrations of 10, 100, and 1000 times the environmental concentration. Joint toxicity experiments evaluated the combined effects of these compounds. Toxic effects were examined during zebrafish embryonic development, and the parameters analyzed were apical sublethal, teratogenicity, mortality endpoints, and morphometry. Thyroid hormone exhibited the highest toxicity. However, the results showed that the environmental concentrations for all 3 compounds had low risk in relation to the parameters studied, such as teratogenic effects and morphometry. The larvae were more affected than embryos, where embryos needed higher concentrations in all experiments, possibly due to the absence of the chorion. The same type of effects were observed in the joint toxicity test, except that a possible antagonistic effect was detected. However, high concentrations showed stronger effects of these toxic compounds on fish development.

Testosterone nanoemulsion produced masculinized Nile tilapia (Oreochromis niloticus).

The objective of this work was to develop a food additive for the sex reversal of Nile tilapia (Oreochromis niloticus) based on a simple oil in water (O/W) nanoemulsion with testosterone propionate for incorporation into commercial feed. Oil screening and evaluation of the organoleptic and physicochemical characteristics were carried out to determine the best formulation. A palatability test was also performed. Sex reversal test was assayed using 5 experimental groups: negative control - macerated feed without hormone; free testosterone - macerated feed with 60 mg/kg of testosterone propionate diluted in ethanol; and macerated feed with testosterone propionate nanoemulsion at a concentration of 30, 60, and 90 mg/kg. Stable nanoemulsions (size 76-210 nm) with testosterone propionate were produced. All nanoemulsion-added feed was palatable to tilapia. We obtained sex reversal values of ≈65, 75, and 72% in the groups of 30, 60, and 90 mg/kg, respectively. We can conclude that the nanoemulsion showed promising results; it is capable of inducing sex reversal in tilapia, is suitable as a commercial product, and has the potential to promote safety for rural staff and reduce the environmental impact of hormones.

Marijuana and Opioid Use during Pregnancy: Using Zebrafish to Gain Understanding of Congenital Anomalies Caused by Drug Exposure during Development.

Marijuana and opioid addictions have increased alarmingly in recent decades, especially in the United States, posing threats to society. When the drug user is a pregnant mother, there is a serious risk to the developing baby. Congenital anomalies are associated with prenatal exposure to marijuana and opioids. Here, we summarize the current data on the prevalence of marijuana and opioid use among the people of the United States, particularly pregnant mothers. We also summarize the current zebrafish studies used to model and understand the effects of these drug exposures during development and to understand the behavioral changes after exposure. Zebrafish experiments recapitulate the drug effects seen in human addicts and the birth defects seen in human babies prenatally exposed to marijuana and opioids. Zebrafish show great potential as an easy and inexpensive model for screening compounds for their ability to mitigate the drug effects, which could lead to new therapeutics.

Folic acid reduces the ethanol-induced morphological and behavioral defects in embryonic and larval zebrafish (Danio rerio) as a model for fetal alcohol spectrum disorder (FASD).

The objective of this work was to determine whether folic acid (FA) reduces the embryonic ethanol (EtOH) exposure induced behavioral and morphological defects in our zebrafish fetal alcohol spectrum disorder (FASD) model. Teratogenic effects, mortality, the excitatory light-dark locomotion (ELD), sleep (SL), thigmotaxis (TH), touch sensitivity (TS), and optomotor response (OMR) tests were evaluated in larvae (6-7 days post-fertilization) using four treatment conditions: Untreated, FA, EtOH and EtOH + FA. FA reduced morphological defects on heart, eyes and swim bladder inflation seen in EtOH exposed fish. The larvae were more active in the dark than in light conditions, and EtOH reduced the swimming activity in the ELD test. EtOH affected the sleep pattern, inducing several arousal periods and increasing inactivity in zebrafish. FA reduces these toxic effects and produced more consistent inactivity during the night, reducing the arousal periods. FA also prevented the EtOH-induced defects in thigmotaxis and optomotor response of the larvae. We conclude that in this FASD model, EtOH exposure produced several teratogenic and behavioral defects, FA reduced, but did not totally prevent, these defects. Understanding of EtOH-induced behavioral defects could help to identify new therapeutic or prevention strategies for FASD.

Protective effects of quercetin, polydatin, and folic acid and their mixtures in a zebrafish (Danio rerio) fetal alcohol spectrum disorder model.

Protective effects of quercetin (QUE), polydatin (POL), and folic acid (FA) and their mixtures were tested using zebrafish to model fetal alcohol spectrum disorder in this study. Zebrafish embryos were exposed to 150 mM ethanol for 6 or 22 h and co-treated with QUE, POL, FA, and their mixtures (37.5-100.0 µM). Epiboly progression, teratogenic effects, and behavior were evaluated. Ethanol exposure reduced epiboly, and FA and QUE protected against these ethanol-induced defects. POL did not reduce epiboly defects. The mixture QUE + FA showed a possible antagonistic effect. The observed teratogenic effects were similar in all ethanol exposed groups. QUE, FA and QUE + POL reduced the percentage of affected animals, but treatments did not eliminate teratogenic effects. Behavioral measurements were divided into small (between 4 and 8 mm/s) and high swimming activity (>8 mm/s). All experimental groups displayed a reduction in small swimming activity as compared to control and ethanol groups when exposed to bright light. Additionally, larvae exposed to ethanol were more inhibited than control, not showing a habituation period (after 60 min of experiment) in high swimming activity. Chemical treatments like QUE and POL reduced behavioral defects induced by ethanol exposure. In conclusion, this study presents new evidence that QUE, POL, FA and their mixtures partially protected epiboly, teratogenic, and behavioral defects induced by ethanol exposure. QUE, FA and QUE + POL were more effective in reducing these defects than the other studied compounds and mixtures.

Values of plasma total proteins of free-living marmosets (Callithrix jacchus) in an urban forest.

BACKGROUND: Callithrix jacchus is a small primate widely used in experimentation, but data on plasma total protein (PTP) values of free-living animals inhabiting its own endemism region are non-existent. METHODS: Marmosets belonging to two free-living groups were captured for marking and obtaining biological material. The collected blood was centrifuged to obtain the plasma and thus to determine the concentration of PTP. RESULTS AND CONCLUSIONS: Females and adults had the highest mean PTP. Mean found were higher than other values described for other neotropical primates, and the high PTP values presented by the animals indicate dehydration. Due to the importance of total plasma proteins for health and management, obtaining these values as a reference is relevant for the free-living Callithrix genus and other neotropical primates.

Thyroxine, levothyroxine, and thyroxine complexed into cyclodextrin changed animal behavior, oxygen consumption, and photopic electroretinogram of Colossoma macropomum.

The toxic effects of thyroxine (T4F), levothyroxine (L-T4), and thyroxine complexed into ß-cyclodextrin (ß-CD-T4) on the biological parameters of tambaqui (Colossoma macropomum) were evaluated. The animals were exposed to a chronic toxicity test based on concentrations of influent (60 ng/L) for 2 months. Weight, total length, animal behavior, oxygen consumption, photopic electroretinogram (ERG), and the Flicker exam were evaluated. No significant differences were observed (p > 0.05) on the weight and total length measurements between all groups studied. Behavioral observations of the animals exposed to L-T4 and ß-CD-T4 complex showed a reduction (p < 0.05) in slow swimming and an increase in staying motionless events. The animals exposed to the ß-CD-T4 complex presented the highest O2 consumption. L-T4 and ß-CD-T4 promoted a reduction in the ability of the animals to respond to stimuli in the photoreceptors according to the photopic ERG examination. Data from the experimental Flicker exam showed no significant differences (p > 0.05) in all groups studied. It can be concluded that the complexation of T4 into ß-CD and L-T4 modified the toxicity of this hormone, promoting changes in the behavior, oxygen consumption, and electrophysiological responses of the exposed animals, suggesting that inclusion complexes should be submitted to new toxicity tests to ensure higher safety.

The complexation of steroid hormones into cyclodextrin alters the toxic effects on the biological parameters of zebrafish (Danio rerio).

The objective of this study was to evaluate the toxic effects of progesterone (P4F) and estradiol (E2F) and the effect of these steroid hormones complexed into cyclodextrins, commercially available drugs, such as micronized progesterone (P4M) and transdermal estradiol (E2T), and evaluate them as endocrine disruptors through biological parameters of Danio rerio. An acute toxicity test was performed with hormones using D. rerio embryos according to OECD 236 guidelines. The heart rate, mortality, and teratogenic effects were evaluated. In addition, a chronic toxicity test was assayed with adult animals for evaluation of animal behavior, reproductive capacity, and electrophysiological responses of the retina. Analysis of the results of the acute toxicity test with embryos exposed to progestins and estrogens showed that free hormones caused a higher percentage of teratogenic effects such as pericardial edema, yolk sac edema, and spinal deformation. Behavioral evaluation (30-60 days) of adult animals exposed to P4M, E2F, and E2T demonstrated higher frequencies of aggressive behaviors such as Chase away, Persecution, Escape, and Attack. Analysis of reproductive capacity did not show significant differences in the number of viable eggs, and no significant changes were observed in the electrophysiological responses of the retina. According to these results, there is a higher toxicity effect of hormones in the free form when compared to the commercial forms and inclusion complexes. This indicates that complexation into cyclodextrin reduced the toxicity of the hormones according to the parameters studied.

Evaluation of the toxic effect of endocrine disruptor Bisphenol A (BPA) in the acute and chronic toxicity tests with Pomacea lineata gastropod.

Bisphenol A (BPA) is a plasticizer and a risk when it interacts with organisms, and can cause changes in the development and reproduction of them. This study aimed to evaluate the effects of BPA, by acute and chronic toxicity tests with neonates and adults of Pomacea lineata. Adults and neonates were divided into groups exposed to BPA (1-20mg/L), or 17ß-estradiol (1mg/L) and control in the acute and chronic toxicity tests. Behavior, heart rate, reproduction and hemolymph biochemical analysis were measured. In the acute toxicity test, the 96-h LC50 with adults was 11.09 and with neonates was 3.14mg/L. In this test, it was observed lethargic behavior and an increase of 77.6% of aspartate aminotransferase in the adults' hemolymph (p<0.05); and neonates' heart rate decreased 72.7% (p<0.05). In the chronic toxicity test, it was observed behaviors associated with reproduction, as Copulate, in the groups exposed to BPA. The results that were found in this study proved that BPA is a potentially toxic agent to Pomacea lineata according to biological parameters evaluated. These data contribute to the understanding of BPA toxic effects' in the aquatic invertebrates.

Cationic liposomes containing antioxidants reduces pulmonary injury in experimental model of sepsis: Liposomes antioxidants reduces pulmonary damage.

The intracellular redox state of alveolar cells is a determining factor for tolerance to oxidative and pro-inflammatory stresses. This study investigated the effects of intratracheal co-administration of antioxidants encapsulated in liposomes on the lungs of rats subjected to sepsis. For this, male rats subjected to sepsis induced by lipopolysaccharide from Escherichia coli or placebo operation were treated (intratracheally) with antibiotic, 0.9% saline and antioxidants encapsulated or non-encapsulated in liposomes. Experimental model of sepsis by cecal ligation and puncture (CLP) was performed in order to expose the cecum. The cecum was then gently squeezed to extrude a small amount of feces from the perforation site. As an index of oxidative damage, superoxide anions, lipid peroxidation, protein carbonyls, catalase activity, nitrates/nitrites, cell viability and mortality rate were measured. Infected animals treated with antibiotic plus antioxidants encapsulated in liposomes showed reduced levels of superoxide anion (54% or 7.650±1.263 nmol/min/mg protein), lipid peroxidation (33% or 0.117±0.041 nmol/mg protein), protein carbonyl (57% or 0.039 ± 0.022 nmol/mg protein) and mortality rate (3.3%), p value <0.001. This treatment also reduced the level of nitrite/nitrate and increased cell viability (90.7%) of alveolar macrophages. Taken togheter, theses results support that cationic liposomes containing antioxidants should be explored as coadjuvants in the treatment of pulmonary oxidative damage.

Glutamine-Loaded Liposomes: Preliminary Investigation, Characterization, and Evaluation of Neutrophil Viability.

Glutamine has received attention due to its ability to ameliorate the immune system response. Once conventional liposomes are readily recognized and captured by immune system cells, the encapsulation of glutamine into those nanosystems could be an alternative to reduce glutamine dosage and target then to neutrophils. Our goals were to nanoencapsulate glutamine into conventional liposomes (Gln-L), develop an analytical high-performance liquid chromatography (HPLC) method for its quantification, and evaluate the viability of neutrophils treated with Gln-L. Liposomes were prepared using the thin-film hydration technique followed by sonication and characterized according to pH, mean size, zeta potential, and drug encapsulation efficiency (EE%). We also aimed to study the effect of liposomal constituent concentrations on liposomal characteristics. The viability of neutrophils was assessed using flow cytometry after intraperitoneal administration of free glutamine (Gln), Gln-L, unloaded-liposome (UL), and saline solution as control (C) in healthy Wistar rats. The selected liposomal formulation had a mean vesicle size of 114.65 ± 1.82 nm with a polydispersity index of 0.30 ± 0.00, a positive surface charge of 36.30 ± 1.38 mV, and an EE% of 39.49 ± 0.74%. The developed chromatographic method was efficient for the quantification of encapsulated glutamine, with a retention time at 3.8 min. A greater viability was observed in the group treated with glutamine encapsulated compared to the control group (17%), although neutrophils remain viable in all groups. Thus, glutamine encapsulated into liposomes was able to increase the number of viable neutrophils at low doses, thereby representing a promising strategy for the treatment of immunodeficiency conditions.