Student perceived impact of a physical, kinetic and interactive model.

Current instructional tools for anatomy education are insufficient scaffolds for learning abstract visual concepts such as basic biomechanics. Diagrams, illustrations and even computer-generated models may be adequate for identification purposes, but are typically not effective when it comes to demonstrating dynamic functions. Evidence from educational concepts, such as the active learning principle in constructivism, indicate that current tools are ineffective due to a lack of opportunity for hands-on or interactive, learning. To address this, our interdisciplinary team designed an instructional model of the canine thoracic limb which is physical, kinetic and interactive. We hypothesised that the unique combination of these three crucial elements would enhance the student learning experience. In order to test this hypothesis, we asked 126 undergraduate students in a biomedical anatomy course to interact with the model. Students were then invited to complete a survey evaluating their experience using the thoracic limb model. Results from completed surveys showed that students perceived the model as easy to use and helpful with understanding basic biomechanical concepts. These results indicate that there is potential for the model to positively impact the students' learning. Future studies will include quantitative evaluation of student performance and cognitive impact in the biomedical anatomy course.

Effects of a novel pesticide-particle conjugate on viability and reactive oxygen species generation in neuronal (PC12) cells.

Development of new methods and compounds to eradicate insect vectors are desperately needed. To that end, our team has previously described the synthesis and characterization of a conjugate comprised of a silver nanoparticle core encapsulated by the pyrethroid pesticide, deltamethrin (pesticide encapsulated silver nanoparticle termed "PENS"). For this current work, the PENS conjugate was tested in neuronal cultured cells to compare the cytotoxic responses to the unconjugated pesticide deltamethrin - a known neurotoxic agent and pristine silver nanoparticles. The PC12 (pheochromocytoma of the rat adrenal medulla) cell line was chosen as a model neuronal culture system. Cells were exposed to known concentrations of PENS, deltamethrin or silver nanoparticle suspensions to assess the degree of toxicity in vitro. After 24 hours of incubation, cell viability and intracellular reactive oxygen species (ROS) were measured. Bright field images of high dose exposures to dosing solutions were also acquired to evaluate cell morphology. Exposure to PENS resulted in a 17% decline in viability at the highest concentration of 45 µM while exposure to deltamethrin caused a 47% decrease. These results suggest that cellular viability was less adversely affected by PENS than by the deltamethrin. Also, ROS production following PENS exposure indicated that the newly developed conjugate was responding in a similar manner as that of cells treated with deltamethrin only.

Surface functionalization of silver nanoparticles: novel applications for insect vector control.

Every day, people and animals contract debilitating and life threatening diseases due to bites from infected flies, ticks, and mosquitoes. The current methods utilized to fight against these diseases are only partially effective or safe for humans and animals. When it comes to insect vector control, a conceptual paradigm shift is urgently needed. This work proposes a novel synthetic scheme to produce a nanoparticle-pesticide core-shell conjugate to be used as an active agent against arthropod vectors, such as mosquitoes. As a proof of concept, we conjugated nanosilver to the pyrethroid pesticide deltamethrin. First, electron microscopy and Fourier transform infrared spectroscopy verified the presence of a 15 nm nanosilver core surrounded by deltamethrin. Second, when the conjugate was exposed to mosquitoes for a 24 h bioassay, mortality was observed at 9 × 10(-4) M. Silver was detected in the hemolymph of mosquitoes exposed to the conjugate. We concluded that the newly developed nanoconjugate did not inactivate the primary function of the pesticide and was effective in killing mosquitoes at low concentrations. These results demonstrate the potential to use nanoparticle surfaces to kill insects, specifically vectors of human pathogens.

The pyrethroid metabolites 3-phenoxybenzoic acid and 3-phenoxybenzyl alcohol do not exhibit estrogenic activity in the MCF-7 human breast carcinoma cell line or Sprague-Dawley rats.

Synthetic pyrethroids are one of the most frequently and widely used class of insecticides, primarily because they have a higher insect to mammalian toxicity ratio than organochlorines or organophosphates. The basic structure of pyrethroids can be characterized as an acid joined to an alcohol by an ester bond. Pyrethroid degradation occurs through either oxidation at one or more sites located in the alcohol or acid moieties or hydrolysis at the central ester bond, the latter reaction being important for mammalian metabolism of most pyrethroids. The primary alcohol liberated from the ester cleavage is hydroxylated to 3-phenoxybenzyl alcohol, which for most pyrethroids is then oxidized to 3-phenoxybenzoic acid. These products may then be conjugated with amino acids, sulfates, sugars, or sugar acids. In vitro studies have suggested that some of the pyrethroids may have estrogenic activity. Interestingly, the chemical structure of specific pyrethroid metabolites indicates that they may be more likely to interact with the estrogen receptor than the parent compounds. Two of the pyrethroid metabolites, 3-phenoxybenzoic acid (3PBA) and 3-phenoxybenzyl alcohol (3PBalc) have been reported to have endocrine activity using a yeast based assay. 3PBAlc exhibited estrogenic activity with reported EC(50)s of 6.67 x 10(-6) and 2 x 10(-5) while 3PBAcid exhibited anti-estrogenic activity with a calculated IC(50) of 6.5 x 10(-5). To determine if the metabolites were able to cause the same effects in a mammalian system, the estrogen-dependent cell line, MCF-7, was utilized. Cells were treated with 1.0, 10.0 or 100.0 microM concentrations of each metabolite and cytotoxicity was assessed. The two lowest concentrations of both metabolites did not induce cell death and even appeared to increase proliferation over that of the control cells. However, when cellular proliferation was measured using a Coulter counter neither metabolite stimulated proliferation (1.0 nM, 10.0 nM, or 10.0 microM) or induced an estrogen receptor alpha/ERE-controlled luciferase reporter in the MCF-7 cells. Following the in vitro screenings, the metabolites were then evaluated for estrogenic activity in vivo using the uterotrophic assay in Sprague-Dawley rats. Animals were orally gavaged (10.0, 5.0, and 1.0 mg/kg) once daily for 3 days. Neither metabolite had any effect on uterine wet weight, body weight, or organ weight. Lastly, in order to determine if either metabolite was able to alter the onset of puberty, immature female rats were orally gavaged (10.0, 5.0, and 1.0 mg/kg) once a day with the metabolites beginning 1 day post-weaning until the onset of puberty as evidenced by vaginal opening (VO). Again, neither metabolite had any effect on the onset of VO.

Insulin-like growth factor-I activates KiSS-1 gene expression in the brain of the prepubertal female rat.

KiSS-1 gene expression has been shown to increase as puberty approaches, and its peptide products, kisspeptins, are involved in LHRH secretion at puberty. Factors contributing to increased KiSS-1 expression, however, have not been identified; thus, the purpose of this study was to assess whether IGF-I could induce transcription of this gene in prepubertal female rats. IGF-I or saline was centrally administered to immature rats that were killed 2, 4, and 6 h later. Real-time PCR revealed that IGF-I induced (P < 0.01) KiSS-1 gene expression at 6 h in a tissue fragment that contained both the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei. Subsequently, the AVPV and ARC nuclei were separated to assess whether region-specific effects could be identified. IGF-I stimulated (P < 0.01) KiSS-1 gene expression in the AVPV nucleus at 6 h after injection, with no change observed in the ARC nucleus. Serum estradiol (E2) levels were not altered at any time point after IGF-I, demonstrating that the increased KiSS-1 expression observed was not caused by an elevation in E2. Additionally, the IGF-I action to induce KiSS-1 gene expression in the AVPV nucleus was further demonstrated when the IGF-I was administered systemically. E2 appears to play an important permissive role because 1-d ovariectomized rats responded to IGF-I with increased (P < 0.01) KiSS-1 expression, whereas, 20 d after ovariectomy, when the E2 levels had fallen below assay sensitivity, the IGF-I was unable to induce KiSS-1 expression. The IGF-I effect was further demonstrated by showing that the IGF-I receptor antagonist, JB-1, blocked the IGF-I-induced increase in KiSS-1 expression. Collectively, these data indicate that IGF-I is an activator of the KiSS-1 gene in the prepubertal female rat.

The pyrethroid pesticide esfenvalerate suppresses the afternoon rise of luteinizing hormone and delays puberty in female rats.

BACKGROUND: One of the most widely used classes of insecticides is the synthetic pyrethroids. Although pyrethroids are less acutely toxic to humans than to insects, in vitro studies have suggested that pyrethroids may be estrogenic. OBJECTIVES: We assessed pubertal effects by orally administering 0.5, 1.0, and 5.0 mg/kg/day of the type II pyrethroid esfenvalerate (ESF) to female rats beginning on postnatal day (PND) 22 until vaginal opening. ESF administration suppresses serum estradiol and delays pubertal onset. MATERIALS AND METHODS: To assess possible hypothalamic and/or pituitary effects, animals received 0.5 or 1.0 mg/kg ESF or corn oil on PNDs 22-29. On PND30, we drew three blood samples (200 microL) from each rat at 15-min intervals beginning at 1000 hours, and again at 1500 hours. To test hypothalamic responsiveness, after the third afternoon sample, all animals received an intravenous injection of N-methyl-d,l-aspartic acid (NMA; 40 mg/kg), and then we drew two more samples. We performed a second experiment as above except that animals received luteinizing hormone-releasing hormone (LHRH; 25 ng/rat) to test pituitary responsiveness. RESULTS: Basal levels of luteinizing hormone (LH) in the afternoon hours were higher in control animals than in animals treated with 1.0 mg/kg ESF (p < 0.05). Furthermore, NMA- and LHRH-stimulated LH release was similar in control and ESF-treated animals, indicating that both hypothalamic and pituitary responsiveness, respectively, were unaffected. CONCLUSIONS: Although the hypothalamus is able to respond to exogenous stimuli, absence of a normal afternoon rise in LH would indicate a hypothalamic deficit in ESF-treated animals.

Comparison of reactive oxygen scavenging systems between a cetacean (DKN1) and a porcine renal epithelial cell line (LLC-PK1).

A transformed renal epithelial cell line, (DKN(1)), from an Atlantic Bottlenose Dolphin, Tursiops truncatus was established in this laboratory and has been used for in vitro genomic analysis and initial toxicological evaluations of dolphin cells. Studies were initiated to compare maintenance of normal antioxidant mechanisms in DKN(1) with similar mechanisms in cells of a pig kidney line, LLC-PK(1). Levels of catalase, glutathione peroxidase, and of reduced glutathione in these dolphin cells were significantly lower than in the porcine cells. Both cell lines were then challenged with hydrogen peroxide at 0.01, 0.1, and 1.0 mM concentrations. The dolphin cells exhibited increased cytotoxicity with a concurrent increase in apoptosis at lower concentrations (0.1 mM) than those required to initiate cytotoxicity in the porcine cells (1.0 mM). Taken together, these results would indicate that the dolphin cells are more susceptible to the damaging effects of certain reactive oxygen species than their terrestrial counterparts.

Effects of alcohol on intraovarian nitric oxide synthase and steroidogenic acute regulatory protein in the prepubertal female rhesus monkey.

OBJECTIVE: In addition to affecting hypothalamic-pituitary function, alcohol is a gonadal toxin capable of inhibiting ovarian function and suppressing circulating levels of estradiol (E2) in female rats, rhesus monkeys, and adolescent girls. Both nitric oxide (NO) and steroidogenic acute regulatory protein (StAR) are intraovarian substances that influence steroidogenesis in opposite directions. This study was undertaken to determine whether alcohol exposure affects prepubertal ovarian steroidogenesis in female rhesus monkeys by altering nitric oxide synthase (NOS), StAR, or both. METHOD: At 20 months of age, monkeys received a single intragastric dose of alcohol (2.4 g/kg) or an equal volume of a saline/sucrose solution daily until they were 36 months old. Blood and ovaries were then collected for assessment of serum hormone levels and tissue gene and protein expression. RESULTS: Alcohol caused depressed levels of serum E2 (p < .05) and luteinizing hormone (p < .05) but not follicle-stimulating hormone. Real-time polymerase chain reaction (RT-PCR) assessment of ovarian mRNA encoding the three isoforms (i.e., neuronal [n] NOS, endothelial [e] NOS, and inducible [i] NOS) of NOS revealed that alcohol exposure did not alter gene expression of nNOS but caused increased basal levels of eNOS (p < .05) and iNOS (p < .01) mRNA expression compared with control ovaries. Alcohol also increased expression of eNOS (p < .01) and iNOS (p < .05) proteins. In contrast, ovaries from monkeys exposed to alcohol showed decreased (p < .05) StAR gene expression compared with controls. CONCLUSIONS: We showed previously that alcohol exposure during adolescence suppressed E2 and delayed development of regular monthly menstruation patterns in rhesus monkeys. The present results suggest that the combined action of alcohol to elevate ovarian NOS and suppress StAR synthesis contributes to these abnormalities.

Manganese stimulates luteinizing hormone releasing hormone secretion in prepubertal female rats: hypothalamic site and mechanism of action.

We have shown recently that Mn2+ stimulates gonadotropin secretion via an action at the hypothalamic level, and a diet supplemented with a low dose of the element is capable of advancing the time of female puberty. In this study, we used an in vitro approach to investigate the mechanism by which Mn2+ induces luteinizing hormone-releasing hormone (LHRH) secretion from prepubertal female rats. The medial basal hypothalamus from 30-day-old rats was incubated in Locke solution for 30 min to assess basal LHRH secretion, then incubated with buffer alone or buffer plus either a nitric oxide synthase (NOS) inhibitor (N-monomethyl-L-arginine (NMMA); 300 or 500 microM) or a soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ); 100 or 250 microM) for another 30 min. Finally, the incubation continued for a further 30 min, but in the presence of MnCl2 (50 or 250 microM) to assess the effect of the blockers on stimulated LHRH secretion. Both 50 and 250 microM MnCl2 stimulated LHRH release (P < 0.05 and P < 0.01, respectively). The addition of 300-500 microM NMMA to the medium did not block Mn2+-stimulated release of LHRH, even with the higher dose of MnCl2. Furthermore, while 50, 100 and 250 microM MnCl2 all significantly induced LHRH release, the two lowest doses did not stimulate total nitrite released from the same tissue, an effect only observed with the highest dose. Taken together, these data suggest that Mn2+ is not an effective stimulator of NO. Conversely, inhibiting sGC with ODQ blocked the Mn2+-stimulated secretion of LHRH in a dose-dependent manner, indicating that GC is the site of action of Mn2+. Additionally, we showed that Mn2+ stimulated cGMP and LHRH from the same tissues, and that downstream blocking of protein kinase G formation with KT5823 (10 microM) inhibited Mn2+-induced LHRH release. These data demonstrate that the principal action of Mn2+ within the hypothalamus is to activate sGC directly and/or as a cofactor with available NO, hence generating cGMP and resulting in prepubertal LHRH release.

Manganese acts centrally to activate reproductive hormone secretion and pubertal development in male rats.

Manganese (Mn) is an important element for normal growth and reproduction. Because Mn accumulates in the hypothalamus and is capable of stimulating puberty-related hormones in female rats, we assessed whether this metal could cause similar effects in male rats. We have demonstrated that MnCl2, when administered acutely into the third ventricle of the brain, acts dose dependently to stimulate luteinizing hormone (LH) release. Furthermore, there was a dose dependent stimulation in the secretion of LH-releasing hormone (LHRH) from the medial basal hypothalamus in vitro, and administration of an LHRH receptor antagonist in vivo blocks Mn-induced LH release. To assess potential chronic effects of the metal, male pups were supplemented with 10 or 25 mg MnCl2 per kg by gastric gavage from day 15 until days 48 or 55, at which times developmental signs of spermatogenesis were assessed. Results demonstrate that while significant effects were not observed with the 10 mg/kg dose, the animals receiving the 25 mg/kg dose showed increased LH (p<0.05), FSH (p<0.01) and testosterone (p<0.01) levels at 55 days of age. Furthermore, there was a concomitant increase in both daily sperm production (p<0.05) and efficiency of spermatogenesis (p<0.05), demonstrating a Mn-induced acceleration in spermatogenesis. Our results suggest Mn is a stimulator of prepubertal LHRH/LH secretion and may facilitate the normal onset of male puberty. These data also suggest that the metal may contribute to male precocious pubertal development should an individual be exposed to low but elevated levels of Mn too early in life.

IGF-1 administration to prepubertal female rats can overcome delayed puberty caused by maternal Pb exposure.

Because prepubertal female rats maternally exposed to lead (Pb) exhibit suppressed serum levels of insulin-like growth factor-1 (IGF-1) and delayed puberty, we investigated the ability of centrally administered IGF-1 to stimulate luteinizing hormone (LH) release in vivo and LH-releasing hormone (LHRH) release in vitro from maternally Pb-exposed prepubertal female rats. Additionally, we assessed whether IGF-1 replacement could affect the timing of female puberty. Results demonstrated that IGF-1 stimulated significantly LH release in both control and Pb-exposed animals. When median eminences from control and Pb-exposed females were incubated with rat IGF-1 in vitro, they responded similarly with significant peptide-induced LHRH release. Lastly, we showed IGF-1 replacement reversed the delay in puberty caused by Pb. These results indicate the central LHRH response to IGF-1 is intact and that Pb-induced delayed puberty is due, at least in part, to suppressed circulating IGF-1 available to the hypothalamus.

Manganese acts centrally to stimulate luteinizing hormone secretion: a potential influence on female pubertal development.

Manganese (Mn), an essential element considered important for normal growth and reproduction, has been shown in adults to be detrimental to reproductive function when elevated. Because Mn can cross the blood-brain barrier and accumulate in the hypothalamus, and because it has been suggested that infants and children are potentially more sensitive to Mn than adults, we wanted to determine the effects of Mn exposure on puberty-related hormones and the onset of female puberty. We demonstrated that MnCl(2) when administered acutely into the third ventricle of the brain acts dose-dependently to stimulate luteinizing hormone (LH) release in prepubertal female rats. Incubation of hypothalami in vitro showed that this effect was due to a Mn-induced stimulation of luteinizing hormone releasing hormone (LHRH). Further demonstration that this is a hypothalamic site of action was shown by in vivo blockade of LHRH receptors and lack of a direct pituitary action of Mn to stimulate LH in vitro. To assess potential short-term effects, animals were supplemented with MnCl(2) (10 mg/kg) by gastric gavage from day 12 until day 29, or, in other animals, until vaginal opening (VO). Mn caused elevated serum levels of LH, follicle stimulating hormone, and estradiol, and it initiated a moderate but significant advancement in age at VO. Our results are the first to show that Mn can stimulate specific puberty-related hormones and suggest that it may facilitate the normal onset of puberty. They also suggest that Mn may contribute to precocious puberty if an individual is exposed to elevated levels of Mn too early in development.