MicroRNA Expression Profiling in Porcine Liver, Jejunum and Serum upon Dietary DON Exposure Reveals Candidate Toxicity Biomarkers.

Deoxynivalenol (DON), a frequent mycotoxin worldwide, impairs human and animal health. The response of microRNAs, small non-coding RNAs, to DON has been scarcely investigated, but holds remarkable potential for biomarker applications. Hence, we aimed to investigate DON-induced changes in the microRNA expression in porcine liver, jejunum and serum by combining targeted and untargeted analyses. Piglets received uncontaminated feed or feed containing 900 µg/kg and 2500 µg/kg DON for four weeks, followed by a wash-out period. In tissue, only slight changes in microRNA expression were detected, with ssc-miR-10b being downregulated in liver of DON-exposed piglets. In serum, several microRNAs were differentially expressed upon DON exposure, four of which were validated by qPCR (ssc-miR-16, ssc-miR-128, ssc-miR-451, ssc-miR-205). The serum microRNA response to DON increased over time and declined after removal of contaminated diets. Receiver operating curve analyses for individual microRNAs were significant, and a combination of the four microRNAs increased the predictive capacity for DON exposure. Predicted microRNA target genes showed enrichment of several pathways including PIK3-AKT, Wnt/ß-catenin, and adherens junctions. This study gives, for the first time, a comprehensive view of the porcine microRNA response to DON, providing a basis for future research on microRNAs as biomarkers for mycotoxins.

Assessment of the risk of death of Clarias gariepinus and Oreochromis niloticus pulse-exposed to selected agricultural pesticides.

Aquatic organisms are often exposed briefly to high pesticide concentration. Survival time model was used to study risk of death in C. gariepinus and O. niloticus fingerlings exposed to 24 mg/L atrazine, 42 mg/l mancozeb, 1 mg/L chlorpyrifos and 0.75 µg/L lambda cyhalothrin for 15, 30, 45 and 60 minutes and continuously for 96 hours. Mortality, time-to-death, weight, length, and condition factor of the fingerlings were recorded. Results obtained showed tilapia was more susceptible than catfish to continuous exposure but not pulse exposure. The survival probability of both species was similar when exposed for 15, 30 and 45 minutes (p > 0.05) but differed after 60 minutes (p < 0.05). Risk of death of catfish exposed briefly to atrazine, mancozeb and chlorpyrifos for 60 minutes was similar to 96 hours continuous exposure, same for tilapia exposed to 1 mg/L chlorpyrifos (p > 0.05). Survival probability of tilapia exposed to chlorpyrifos for 15, 30, 45 and 60 minutes was similar (p > 0.05) and was not influenced by pulse length. Pesticide hazard and risk of death decreased as fish size (weight, length, and condition factor) increased. Pulse toxicity assessment using survival models could make pesticides exposure assessment more realistic by studying factors that can influence the toxicity of pesticides.

Fish early life stage toxicity prediction from acute daphnid toxicity and quantum chemistry.

One step towards reduced animal testing is the use of in silico screening methods to predict toxicity of chemicals, which requires high-quality data to develop models that are reliable and clearly interpretable. We compiled a large data set of fish early life stage no observed effect concentration endpoints (FELS NOEC) based on published data sources and internal studies, containing data for 338 molecules. Furthermore, we developed a new quantitative structure-activity-activity relationship (QSAAR) model to inform estimation of this endpoint using a combination of dimensionality reduction, regularization, and domain knowledge. In particular, we made use of a sparse partial least squares algorithm (sPLS) to select relevant variables from a huge number of molecular descriptors ranging from topological to quantum chemical properties. The final QSAAR model is of low complexity, consisting of 2 latent variables based on 8 molecular descriptors and experimental Daphnia magna acute data (EC50, 48 h). We provide a mechanistic interpretation of each model parameter. The model performs well, with a coefficient of determination r 2 of 0.723 on the training set (cross-validated q 2 = 0.686) and comparable predictivity on a test data set of chemically related molecules with experimental Daphnia magna data (r 2 test = 0.687, RMSE = 0.793 log units).

Organochlorine pesticides: Agrochemicals with potent endocrine-disrupting properties in fish.

Organochlorine pesticides (OCPs) are persistent environmental contaminants that act as endocrine disruptors and organ system toxicants. These pesticides (e.g. dichlorodiphenyltrichloroethane (DDT), dieldrin, toxaphene, among others) are ranked as some of the most concerning chemicals for human health. These pesticides (1) act as teratogens, (2) are neuroendocrine disruptors, (3) suppress the immune and reproductive systems, and (4) dysregulate lipids and metabolism. Using a computational approach, we revealed enriched endocrine-related pathways in the Comparative Toxicogenomics Database sensitive to this chemical class, and these included reproduction (gonadotropins, estradiol, androgen, steroid biosynthesis, oxytocin), thyroid hormone, and insulin. Insight from the Tox21 and ToxCast programs confirm that these agrochemicals activate estrogen receptors, androgen receptors, and retinoic acid receptors with relatively high affinity, although differences exist in their potency. We propose an adverse outcome pathway for OCPs toxicity in the fish testis as a novel contribution to further understanding of OCP-induced toxicity. Organochlorine pesticides, due to their persistence and high toxicity to aquatic and terrestrial wildlife as well as humans, remain significant agrochemicals of concern.

Exposição crônica ao cloridrato de metformina e à glibenclamida causa alterações comportamentais, glicêmicas e de mortalidade em Hemigrammus caudovittatus e Danio rerio / Chronic exposure to metformin chloridrate and glibenclamide causes behavioral, blood glucose and mortality changes of Hemigrammus caudovittatus and Danio rerio

Hemigrammus caudovittatus e Danio rerio foram expostos aos hipoglicemiantes orais (HOs) cloridrato de metformina a 40µg/L e 120µg/L e glibenclamida a 0,13µg/L e 0,39µg/L durante 100 dias. Foram avaliados os efeitos tóxicos dos fármacos em relação ao peso, ao comportamento animal, à glicemia e à mortalidade. H. caudovittatus expostos à menor concentração dos fármacos apresentaram aumento significativo (P<0,05) no evento Respiração Aérea. Ainda, foi observado aumento no comportamento Descansar quando os animais foram expostos à glibenclamida a 0,39µg/L. Em D. rerio expostos ao cloridrato de metformina a 120µg/L, foi observado aumento (P<0,05) no comportamento Descansar. A glibenclamida provocou redução (P<0,05) na glicemia de H. caudovittatus. Ambos os fármacos causaram efeito letal na espécie D. rerio, contudo a glibenclamida foi mais tóxica, causando 100% de mortalidade em 30 dias de exposição. Os animais que vieram a óbito apresentaram congestão nos arcos branquiais e hemorragia. Os HOs foram desenvolvidos para apresentarem efeitos fisiológicos em mamíferos, entretanto efeitos tóxicos foram encontrados nas duas espécies de peixe estudadas. Isso levanta a preocupação sobre possíveis efeitos tóxicos de HOs e sobre quais métodos serão utilizados para a sua degradação no ambiente aquático.(AU)

Hemigrammus caudovittatus and Danio rerio were exposed to oral hypoglycemic drugs (HOs) metformin hydrochloride at 40µg/L and 120µg/L and to glibenclamide at 0.13µg/L and 0.39µg/L during 100 days. Toxic effects of the drugs were evaluated based on weight, animal behavior, blood glucose and mortality. H. caudovittatus exposed to lowest concentration of the drugs showed significant increase (P< 0.05) in the Air breathing event. Furthermore, increase in Rest event was observed when animals were exposed to glibenclamide at 0.39µg/L. An increase (P< 0.05) in the frequency of Rest behavior in the D. rerio exposed to metformin hydrochloride at 120µg/L was observed. Glibenclamide caused decrease (P< 0.05) in the blood glucose of H. caudovittatus. Both drugs caused lethal effect against D. rerio. Nevertheless, glibenclamide was more toxic causing 100% of mortality after 30 days of exposure. The animals that died showed congestion on the branchial arches and hemorrhage. The HOs were developed to have physiological effects in mammals. However, toxic effects were found in both species of fish studied. This raises concerns about possible toxic effects of HOs and what methods will be used for their degradation in the aquatic environment.(AU)

The Contribution of In Vivo Mammalian Studies to the Knowledge of Adverse Effects of Radiofrequency Radiation on Human Health.

The proliferation of cellular antennas and other radiofrequency radiation (RFR) generating devices of the last decades has led to more and more concerns about the potential health effects from RFR exposure. Since the 2011 classification as a possible carcinogen by the International Agency for Research on Cancer (IARC), more experimental studies have been published that support a causal association between RFR exposure and health hazards. As regard cancer risk, two long-term experimental studies have been recently published by the US National Toxicology Program (NTP) and the Italian Ramazzini Institute (RI). Despite important experimental differences, both studies found statistically significant increases in the development of the same type of very rare glial malignant tumors. In addition to carcinogenicity, reproductive organs might be particularly exposed, as well as sensitive to RFR. In this work, we reviewed the currently available evidence from in vivo studies on carcinogenicity and reproductive toxicity studies in order to summarize the contribution of experimental research to the prevention of the adverse effects of RFR on human health.

Incidental induction of secondary bowel disorders in guinea pigs during a batch safety test of veterinary vaccines.

Veterinary vaccines are subjected to a safety testing using laboratory animals via intraperitoneal injection per batch. From April 2010 to March 2011, 7 guinea pigs in 4 batch tests exhibited unrecoverable weight loss and/or were found dead. Six guinea pigs had developed intussusception, whereas another one had developed an intestinal obstruction consequent to adhesion. A histopathology revealed that these lesions were associated with inflammatory foci. Other animals than the 7 guinea pig also developed similar inflammatory foci but did not develop bowel disorders. In the retesting of these batches, animals did not exhibited clinical signs, though inflammatory foci were detected. The clinical signs, detected in the primary test, might be due to bowel disorders secondary to an inflammatory response, rather than toxicity.

Guideline on safety evaluation of cell-based medicinal products for animal use.

With the increased use of cell therapy in the veterinary sector, there is a growing demand for the development of cell-based medicinal products and the determination of their safety. Currently, the Korean Animal and Plant Quarantine Agency has established a guideline for evaluating the safety of cell-based medicinal products for animal use. The guideline includes items related to definition, classification, management, manufacturing procedure and quality control (standard and test method), stability testing, toxicity testing, pharmacological testing, and performance of clinical trials. In addition, testing protocols related to safety assessment of animal cell-based products such as chromosome karyotyping, tumorigenicity testing, confirmatory testing of biodistribution and kinetics, and target animal safety testing are described in detail. Moreover, because cell-based medicinal products are novel therapies, deviations from traditional designs may be justified in order to obtain relevant safety information on the treatment. Additionally, this guideline can be amended on the basis of new scientific findings.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 2 of 2).

To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist's examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions. In exercise #2, there was improved consistency of categorization of small borderline morphologies and mild lesions, but a decrement in consistency of categorizing minimal lesions. Exercises 1 and 2 suggest the described diagnostic approach is representative of that in use by the majority of toxicologic pathologists across companies/agencies and that application by all may improve diagnostic consistency of PCM/like lesions. Additionally, a criterion of approximately 5% heart section involvement is suggested for separating mild from moderate or greater severity. While evidence is not absolute, until further investigation shows otherwise, microscopic changes resembling PCM, but located in the epicardial and subepicardial region of the right ventricle, may be considered as part of the spectrum of PCM.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 1 of 2).

Spontaneous rodent progressive cardiomyopathy (PCM) in the Sprague Dawley rat may confound identification and/or interpretation of potential test article (TA)-related cardiotoxicity. Pathologists apply diagnostic term(s) and thresholds for diagnosing and assigning severity grades for PCM and/or PCM-like (PCM/like) lesions consistently within a study, which is necessary to identify and interpret TA-related findings. Due to differences in training and/or experiences, diagnostic terms and thresholds may vary between pathologists. Harmonized terminology and thresholds across studies will generate better historical control data, will likely enhance interpretation of study data, and may further enhance our understanding of the spontaneous change. An assessment of the diagnostic approaches of a group of 37 pathologists identified an approach that is relatively easily applied; and if adopted, it could enhance diagnostic consistency across studies. This approach uses the single "slash" term "necrosis/inflammatory cell infiltrate (NICI)" as the diagnosis for the spectrum of lesions seen in younger rats, uses no threshold for diagnosis (e.g., diagnose all lesions clearly identifiable as PCM/like), and uses aggregate lesion size of approximately ≥45% of the field of view (FOV) using a 10×/22 eyepiece and the 40× objective or approximately ≥100% of the FOV using the 60× objective as the criterion separating minimal from mild severities.

Deciphering Sources of Variability in Clinical Pathology-It's Not Just about the Numbers.

Preanalytical variables can have significant impacts on clinical pathology parameters evaluated during the conduct of a nonclinical safety or toxicity study. These preanalytical variables can be controlled by careful attention to factors such as animal dietary status (diet composition, fasted, and fed state), restraint and anesthesia, intercurrent procedures, timing of clinical pathology collections, and proficiency of animal technicians. The impact of preanalytical variables on test results can be significant enough to result in difficult interpretations and/or regulatory questions or can obfuscate the effects of a test article. Control of preanalytical variables starts with knowledge of what processes and procedures impact test results. Minimizing these effects improves the quality of results and maximizes the value of the study.

Principles for Assessing Adversity in Toxicologic Clinical Pathology.

There is limited direction in the literature or regulatory guidance on determination of adversity for clinical pathology (CP) biomarkers in preclinical safety studies. Toxicologic clinical pathologists representing the American Society for Veterinary Clinical Pathology-Regulatory Affairs Committee and Society of Toxicologic Pathology-Clinical Pathology Interest Group identified principles, overall approach, and unique considerations for assessing adversity in CP data interpretation to provide a consensus opinion. Emphasized is the need for pathophysiologic context and a weight-of-evidence approach. Most CP biomarkers do not have the potential to be adverse in isolation, regardless of magnitude of change. Rather, they quantify or describe the impact of effects, provide adjunct or supportive information regarding a process or pathogenesis, and provide translational biomarkers of effect. Most often, CP changes are part of a constellation of findings that collectively are adverse. Thus, most CP changes must be interpreted in conjunction with other study findings and require contextual and integrative interpretation. Exceptions include critical CP changes without correlates that indicate a health risk in the tested species. Overall, CP changes should not be interpreted in isolation and their adversity is best addressed with an integrated approach.

Profiling of Selected Functional Metabolites in the Central Nervous System of Marine Medaka (Oryzias melastigma) for Environmental Neurotoxicological Assessments.

The simultaneous profiling of 43 functional metabolites in the brain of the small model vertebrate organism, marine medaka (Oryzais melastigma), has been accomplished via dansyl chloride derivatization and LC-MS/MS quantification. This technique was applied to examine effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), one of the most abundant polybrominated diphenyl ether flame retardants in the natural environment, on the central nervous system (CNS) of vertebrates. The model teleosts were fed with bioencapsulated Artemia nauplii for up to 21 days. Multivariate statistical analysis has demonstrated that levels of numerous classical neurotransmitters and their metabolites in the CNS of the fish were perturbed even at the early phase of dietary exposure. Subsequent metabolic pathway analysis further implied potential impairment of the arginine and proline metabolism; glycine, serine and threonine metabolism; D-glutamine and D-glutamate metabolism; alanine, aspartate, and glutamate metabolism; valine, leucine, and isoleucine biosynthesis, and the cysteine and methionine metabolism in the brain of the test organism. Our results demonstrate that targeted profiling of functional metabolites in the CNS may shed light on how the various neurological pathways of vertebrates, including humans, are affected by toxicant/stress exposure.

Cytological Bone Marrow Cell Differential Counts and Morphologic Findings in Healthy Cynomolgus Monkeys ( Macaca fascicularis) from Nonclinical Toxicology Studies.

Cytological bone marrow evaluation is utilized in nonclinical toxicology studies to characterize hematopoietic effects when the combined interpretation of histologic and complete blood count data does not yield sufficient information. Results from cytological bone marrow examination should be interpreted in the context of variability observed in concurrent control animals with consideration of cytologist experience and historical/published data. Cytological bone marrow differential counts and cellular morphologic findings from 130 (66 male, 64 female) healthy control cynomolgus monkeys from nonclinical toxicology studies were retrospectively analyzed. Myeloid to erythroid (M:E) ratios and the percentage of total cells for each cell type were determined from differential cell count data. M:E ratios ranged from 0.6:1 to 2.3:1. Percentages of total granulocytic cells, total erythroid cells, and lymphocytes ranged from 26.6% to 60.6%, 25.7% to 52.2%, and 5.5% to 40.4%, respectively. Monocytes, plasma cells, mast cells, and mitotic figures were typically <1% of total cells. Notable morphologic findings included occasional giant neutrophilic metamyelocytes and band neutrophils, ring-shaped band neutrophil nuclei, metarubricyte nuclear blebbing and binucleation, multiple or nonfused megakaryocyte nuclei, and emperipolesis. These results represent cytological bone marrow findings from healthy control cynomolgus monkeys utilized in nonclinical toxicology studies and provide insight into expected background variability.

Evaluation of Pepsinogen I as a Biomarker of Drug-induced Gastric Mucosal Injury in Cynomolgus Monkeys.

Gastric mucosal injury is frequently observed in nonclinical studies of nonhuman primates. Because microscopic evaluation of stomach is generally a terminal procedure, our objective was to determine whether serum pepsinogen I (PG I) could serve as a noninvasive biomarker for detection of gastric mucosal injury in monkey. Serum PG I was measured using a commercial human immunoassay in cynomolgus monkeys ( n = 166) prior to dosing and/or terminally in 11 studies of up to 1 month duration. Mean ( SD) PG I values (ug/L) for monkeys with ( n = 59) and without ( n = 100) gastric mucosal degeneration were 101 (215) and 28 (12.6), respectively. For monkeys with baseline and terminal PG I data, mean ( SD) fold change (ratio of terminal to baseline PG I) for monkeys with ( n = 57) and without ( n = 76) glandular degeneration were 4.1 (11.3) and 1 (0.28). Receiver operating characteristic area under the curve (AUC) data demonstrated moderate diagnostic accuracy for serum PG I for glandular degeneration, AUC ( SE) 0.789 (0.04), with improved diagnostic accuracy as a fold change of baseline, AUC ( SE) 0.816 (0.04), consistent with the large interindividual but low intraindividual variability of serum PG I values in control monkeys. These data demonstrate that serum PG I is a useful biomarker of drug-induced gastric mucosal injury in the cynomolgus monkey.

A screening of medicinal compounds for their effect on egg strings and nauplii of the salmon louse Lepeophtheirus salmonis (Krøyer).

Egg strings and nauplii of the salmon louse Lepeophtheirus salmonis were exposed to a variety of medicinal compounds at 50 mg L(-1) for 30 min in two experiment series. This medicine concentration was selected as a starting point for a screening series. Hatching of egg strings and development to copepodid larvae were monitored in one experiment, and the survival and development of nauplii were monitored in the other. Two compounds, emamectin benzoate and cypermethrin, inhibited hatching effectively. Several compounds affected nauplii, either directly or through inhibiting development to the infective stage. A total of 50 mg L(-1) of azamethiphos, cypermethrin, emamectin benzoate and propoxur was lethal to >70% of the larvae. Diflubenzuron, fenoxycarb, pymetrozine, pyriprole and tebufenozide diminished the ability of nauplii developing to copepodids.

Pharmacological characterization of venoms from three theraphosid spiders: Poecilotheria regalis, Ceratogyrus darlingi and Brachypelma epicureanum

Background Tarantulas (Theraphosidae) represent an important source of novel biologically active compounds that target a variety of ion channels and cell receptors in both insects and mammals. In this study, we evaluate and compare the pharmacological activity of venoms from three taxonomically different theraphosid spiders bred in captivity: Poecilotheria regalis, an aggressive arboreal tarantula from southeastern India; Ceratogyrus darlingi, an aggressive tarantula from southern Africa; and Brachypelma epicureanum, a docile tarantula from the Yucatan dry forest of Mexico. Prior to this study, no research had been conducted with regard to the composition and pharmacological activity of these venoms. Methods The pharmacological characterization of the venoms was described for the first time by the assessment of their toxicity in crickets (LD50) along with their nociceptive (by using the formalin test), hyaluronidase, phospholipase A2, edematogenic and caseinolytic activity. Results P. regalis and B. epicureanum venoms induced a similar lethal effect on crickets (LD50 = 5.23 ± 3.1 and 14.4 ± 5.0 μg protein/g 48 h post-injection, respectively), whereas C. darlingi venom (119.4 ± 29.5 μg protein/g 48 h post-injection) was significantly less lethal than the other two venoms. All three venoms induced similar edematogenic activity on rats but did not induce nociceptive behavior. The assessment of enzymatic activity indicated that P. regalis venom induces significantly higher hyaluronidase activity (27.6 ± 0.9 TRU/mg) than both C. darlingi (99.7 ± 1.9 TRU/mg) and B. epicureanum (99.6 ± 1.6 TRU/mg); these latter venoms did not display phospholipase A2or caseinolytic activity. Conclusions This study demonstrates that these theraphosid spiders of different habitats produce venoms with different activities. P. regalis venom displays a high level of hyaluronidase activity, which may be associated with its potentially medically significant bite.

Exposure to 50 Hz electromagnetic field changes the efficiency of the scorpion alpha toxin

Background Extremely low-frequency (50 Hz) electromagnetic field (ELF-EMF) is produced by electric power transmission lines and electronic devices of everyday use. Some phenomena are proposed as first effects of ELF-EMF: the discrete changes in the membrane potential and the increase of the calcium channel activity as well as the intracellular concentration of Ca 2+ . Interaction of the scorpion alpha toxin with the sodium channel depends on the orientation of the charges and may be perturbed by changes in the membrane polarization. The toxin induces overexcitability in the nervous system and an increase in the neurotransmitters released with different consequences, mainly the paralysis of muscles. We assumed that the exposure to ELF-EMF 0.7 mT will change the effects of the insect selective scorpion alpha toxin (recombinant LqhIT from Leiurus quinquestriatus hebraeus) at the level of the cercal nerve function, the synaptic transmission and on the level of entire insect organism. Taking into account the compensatory mechanisms in organisms, we tested in addition ten times higher ELF-EMF on whole insects.Methods Experiments were performed in vivo on cockroaches (Periplaneta americana) and in vitro on isolated cockroach abdominal nerve cord with cerci. In biotests, the effects of LqhIT (10 8 M) were estimated on the basis of the insect ability to turn back from dorsal to ventral side. Three groups were compared: the control one and the two exposed to ELF-EMF 0.7 and 7 mT. Bioelectrical activity of the cercal nerve and of the connective nerve that leaves the terminal abdominal ganglion was recorded using extracellular electrodes. LqhIT (5 × 10 8 M) induced modifications of neuronal activity that were observed in the control cockroach preparations and in the ones exposed to ELF-EMF (0.7 mT). The exposure to ELF-EMF was carried out using coils with a size appropriate to the examined objects.Results The exposure to ELF-EMF (0.7 mT) modified the effects of LqhIT (5 × 108 M) on activity of the cercal nerve and of the connective nerve. We observed a decrease of the toxin effect on the cercal nerve activity, but the toxic effect of LqhIT on the connective nerve was increased. Biotests showed that toxicity of LqhIT (10 8 M) on cockroaches was reduced by the exposure to ELF-EMF (0.7 and 7 mT).Conclusions The exposure to 50 Hz ELF-EMF modified the mode of action of the anti-insect scorpion alpha toxin LqhIT at cellular level of the cockroach nervous system and in biotests. Toxin appeared as a usefull tool in distinguishing between the primary and the secondary effects of ELF-EMF.

Allosteric interactions between receptor site 3 and 4 of voltage-gated sodium channels: a novel perspective for the underlying mechanism of scorpion sting-induced pain

Background BmK I, a site-3-specific modulator of voltage-gated sodium channels (VGSCs), causes pain and hyperalgesia in rats, while BmK IT2, a site-4-specific modulator of VGSCs, suppresses pain-related responses. A stronger pain-related effect has been previously attributed to Buthus martensi Karsch (BmK) venom, which points out the joint pharmacological effect in the crude venom.Methods In order to detect the joint effect of BmK I and BmK IT2 on ND7-23 cells, the membrane current was measured by whole cell recording. BmK I and BmK IT2 were applied successively and jointly, and the synergistic modulations of VGSCs on ND7-23 cells were detected.Results Larger peak I Na and more negative half-activation voltage were elicited by joint application of BmK I and BmK IT2 than by application of BmK I or BmK IT2 alone. Compared to the control, co-applied BmK I and BmK IT2 also significantly prolonged the time constant of inactivation.Conclusions Our results indicated that site-4 toxin (BmK IT2) could enhance the pharmacological effect induced by site-3 toxin (BmK I), suggesting a stronger effect elicited by both toxins that alone usually exhibit opposite pharmacological effects, which is related to the allosteric interaction between receptor site 3 and site 4. Meanwhile, these results may bring a novel perspective for exploring the underlying mechanisms of scorpion sting-induced pain.

Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension.

The development and optimization of preantral follicle culture methods are crucial in fertility preservation strategies. As preantral follicle dynamics are usually assessed by various invasive techniques, the need for alternative noninvasive evaluation tools exists. Recently, neutral red (NR) was put forward to visualize preantral follicles in situ within ovarian cortical fragments. However, intense light exposure of NR-stained tissues can lead to cell death because of increased reactive oxygen species production, which is also associated with elevated oxygen tension. Therefore, we hypothesize that after repeated NR staining, follicle viability and dynamics can be altered by changes in oxygen tension. In the present study, we aim (1) to determine whether NR can be used to repeatedly assess follicular growth, activation, and viability and (2) to assess the effect of a low (5% O2) or high (20% O2) oxygen tension on the viability, growth, and stage transition of preantral follicles cultured in vitro by means of repeated NR staining. Cortical slices (n = 132; six replicates) from bovine ovaries were incubated for 3 hours at 37 °C in a Leibovitz medium with 50 µg/mL NR. NR-stained follicles were evaluated in situ for follicle diameter and morphology. Next, cortical fragments were individually cultured in McCoy's 5A medium for 6 days at 37 °C, 5% CO2, and 5% or 20% O2. On Days 4 and 6, the fragments were restained by adding NR to the McCoy's medium and follicles were reassessed. In both low and high oxygen tension treatment groups, approximately 70% of the initial follicles survived a 6-day in vitro culture, but no significant difference in follicle survival on Day 4 or 6 could be observed compared with Day 0 (P > 0.05). A significant decrease in the number of primordial and increase in primary and secondary follicles was observed within 4 days of culture (P < 0.001). In addition, a significant increase of the mean follicle diameter in NR-stained follicles was observed (P < 0.001), resulting in an average growth of 11.82 ± 0.81 µm (5% O2) and 11.78 ± 1.06 µm (20% O2) on Day 4 and 20.94 ± 1.24 µm (5% O2) and 19.12 ± 1.36 µm (20% O2) on Day 6 compared with Day 0. No significant differences in follicle growth rate or stage transition could be observed between 5% and 20% O2 (P > 0.05). In conclusion, after repeated NR staining, we could not find a difference between low and high oxygen tension in terms of follicle viability, stage transition, or growth. Therefore, under our culture conditions follicle dynamics are not determined by the oxygen tension in combination with quality assessment protocols using repeated NR staining.