Warmer temperature increases toxicokinetic elimination of PCBs and PBDEs in Northern leopard frog larvae (Lithobates pipiens).

We studied the temperature dependence of accumulation and elimination of two polychlorinated biphenyls (PCBs; PCB-70 and PCB-126) and a commercial mixture of congeners of polybrominated diphenyl ethers (PBDEs; DE-71™)) in Northern leopard frog (Lithobates pipiens) tadpoles. We reared tadpoles at 18, 23, or 27 °C for 5.3 or up to 13.6 weeks (longer at cooler temperature where development is slower) on diets containing the toxicants, each at several different toxicant concentrations, and compared tissue concentrations as a function of food concentration and rearing temperature. Following > 1 month of accumulation, tissue concentrations of all three toxicants in exposed tadpoles were linearly related to dietary concentrations as expected for first order kinetics, with no significant effect of rearing temperature.We also raised free-swimming L. pipiens tadpoles for 14 days on foods containing either toxicant at 18 or 27 °C during an accumulation phase, and then during depuration (declining toxicant) phase of 14 days we provided food without toxicants and measured the decline of toxicants in tadpole tissue. All the congeners were eliminated faster at warmer rearing temperature, as expected. Using Arrhenius' equation, we calculated that the apparent activation energy for elimination of both PCB congeners by tadpoles was 1.21 eV (95% confidence interval 0.6-1.8 eV). We discuss how this value was within the range of estimates for metabolic reactions generally (range 0.2 - 1.2 eV), which might include metabolic pathways for biotransformation and elimination of PCBs. Furthermore, we discuss how the lack of an effect of rearing temperature on tadpole near-steady-state tissue residue levels suggests that faster elimination at the warmer temperature was balanced by faster uptake, which is plausible considering the similar temperature sensitivities (i.e., activation energies) of all these processes. Although interactions between toxicants and temperature can be complex and likely toxicant-dependent, it is plausible that patterns observed in tadpoles might apply to other aquatic organisms. Published data on depuration in 11 fish species eliminating 8 other organic toxicants indicated that they also had similar apparent activation energy for elimination (0.82 ± 0.12 eV; 95% confidence interval 0.56 - 1.08 eV), even though none of those studied toxicants were PCBs or PBDEs. Additional research on toxicant-temperature interactions can help improve our ability to predict toxicant bioaccumulation in warming climate scenarios.

Single and mixture per- and polyfluoroalkyl substances accumulate in developing Northern leopard frog brains and produce complex neurotransmission alterations.

Per- and polyfluoroalkyl substances (PFAS) are present in water and >99% of human serum. They are found in brains of wildlife; however, little is known about effects on the developing brain. To determine the effects of PFAS on brain and cardiac innervation, we conducted an outdoor mesocosm experiment with Northern leopard frog larvae (Rana pipiens) exposed to control, 10 ppb perfluorooctane sulfonate (PFOS), or a PFAS mixture totaling 10 ppb that mimicked aqueous film forming foam-impacted surface water (4 ppb PFOS, 3 ppb perfluorohexane sulfonate, 1.25 ppb perfluorooctanoate, 1.25 ppb perfluorohexanoate, and 0.5 ppb perfluoro-n-pentanoate). Water was spiked with PFAS and 25 larvae (Gosner stage (GS) 25) added to each mesocosm (n = 4 mesocosms per treatment). After 30 days, we harvested eight brains per mesocosm and remaining larvae developed to GS 46 (i.e. metamorphosis) before brains and hearts were collected. Weight, length, GS, and time to metamorphosis were recorded. Brain concentrations of all five PFAS were quantified using LC/MS/MS. Dopamine and metabolites, serotonin and its metabolite, norepinephrine, γ-aminobutyric acid, and glutamate were quantified using High Performance Liquid Chromatography with electrochemical detection while acetylcholine and acetylcholinesterase activity were quantified with the Invitrogen Amplex Red Acetylcholine Assay. PFOS accumulated in the brain time- and dose-dependently. After 30 days, the mixture decreased serotonin while both PFAS treatments decreased glutamate. Interestingly, acetylcholine increased in PFAS treatments at GS 46. This research shows that developmental environmentally relevant exposure to PFAS changes neurotransmitters, especially acetylcholine.

Bioevaluation of Ranatuerin-2Pb from the Frog Skin Secretion of Rana pipiens and its Truncated Analogues.

Antimicrobial peptides (AMPs) are considered as a promising agent to overcome the drug-resistance of bacteria. Large numbers of AMPs have been identified from the skin secretion of Rana pipiens, including brevinins, ranatuerins, temporins and esculentins. In this study, the cDNA precursor of a broad-spectrum antimicrobial peptide, ranatuerin-2Pb, was cloned and identified. Additionally, two truncated analogues, RPa and RPb, were synthesised to investigate the structure-activity relationship of ranatuerin-2Pb. RPa lost antimicrobial activity against Candida albicans, MRSA, Enterococcus faecalis and Pseudomonas aeruginosa, while RPb retained its broad-spectrum antimicrobial activity. Additionally, ranatuerin-2Pb, RPa and RPb demonstrated inhibition and eradication effects against Staphylococcusaureus biofilm. RPb showed a rapid bacterial killing manner via membrane permeabilization without damaging the cell membrane of erythrocytes. Moreover, RPb decreased the mortality of S. aureus infected Galleria mellonella larvae. Collectively, our results suggested that RPb may pave a novel way for natural antimicrobial drug design.

Validation of water-borne cortisol and corticosterone in tadpoles: Recovery rate from an acute stressor, repeatability, and evaluating rearing methods.

Amphibian populations are declining globally, so understanding how individuals respond to anthropogenic and environmental stressors may aid conservation efforts. Using a non-invasive water-borne hormone assay, we measured the release rates of two glucocorticoid hormones, corticosterone and cortisol, in Rio Grande Leopard frog, Rana berlandieri, tadpoles. We validated this method pharmacologically and biologically using an adrenocorticotropic hormone (ACTH) challenge, exposure to exogenous corticosterone, and an agitation test. We calculated the repeatability of hormone release rates, the recovery time from an acute stressor, and explored rearing methods for tadpoles. Tadpole corticosterone release rates increased following an ACTH challenge, exposure to exogenous corticosterone, and agitation, validating the use of water-borne hormone methods in this species. After exposure to an acute stressor via agitation, corticosterone release rates began to decline after 2 h and were lowest after 6 h, suggesting a relatively rapid recovery from an acute stressor. Tadpoles reared in groups had higher corticosterone release rates than tadpoles reared individually, and lost mass by Day 7, while tadpoles reared individually did not show a stress response, therefore either rearing method is viable, but have differing physiological costs for tadpoles. Repeatability of corticosterone release rates was moderate to high in R. berlandieri tadpoles, indicating that this species can show a response to selection and potentially respond to rapid environmental change. Our results show that the water-borne hormone assay is a viable way to measure glucocorticoids in this species and is useful in the field of conservation physiology for rare and endangered species.

Effect of Individual and Combined Treatments of Pesticide, Fertilizer, and Salt on Growth and Corticosterone Levels of Larval Southern Leopard Frogs (Lithobates sphenocephala).

Human activities have introduced a variety of chemicals, including pesticides, fertilizers, and salt, into the environment, which may have deleterious effects on the organisms inhabiting these areas. Amphibians are especially susceptible to absorption of chemical pollutants. To determine the possible combined effects of these chemicals on amphibian development and stress levels, Southern leopard frog (Lithobates sphenocephala) larvae were exposed to one of eight individual or combined treatments of atrazine, ammonium nitrate fertilizer, and sodium chloride salt. Stress levels, indicated by release of the stress hormone corticosterone, were measured premetamorphosis at week 8 of development. Water hormone samples were processed to analyze corticosterone levels. Changes in tadpole growth were determined by surface area measurements taken from biweekly photographs. The combined chemical treatment of atrazine, salt, and fertilizer had a significant interactive effect by increasing stress levels before metamorphosis (p = 0.003). After a month of larval development, tadpoles exposed to ammonium nitrate had larger surface area (p = 0.035). Tadpoles exposed to atrazine had a lower growth rate throughout larval development (p = 0.025) and the lowest number of individuals reaching metamorphosis at 33%. However, the frogs in the atrazine treatment that did successfully metamorphose did so in fewer days (p = 0.002). Because amphibians are exposed to multiple chemicals simultaneously in the environment, assessing the effects of a combination of contaminants is necessary to improve application strategies and ecosystem health.

Effect of glucocorticoids on expression of cutaneous antimicrobial peptides in northern leopard frogs (Lithobates pipiens).

BACKGROUND: Many species of frogs secrete cutaneous antimicrobial peptides that are capable of killing Batrachochytrium dendrobatidis. Some of these species are nonetheless susceptible to chytridiomycosis, suggesting that host factors causing dysregulation of this innate immune response may be important in pathogenesis. Since stresses, such as from environmental perturbations, are a potential cause of such dysregulation, this study investigated the effect of glucocorticoid on cutaneous gene expression of these antimicrobial peptides. RESULTS: Northern leopard frogs (Lithobates pipiens) were injected with either the corticosteroid methylprednisolone or saline every 48 h. Norepinephrine-elicited cutaneous secretions were collected every 8 days for 40 days. Gene expression of antimicrobial peptides (brevinin-1P and ranatuerin-2P) in the cutaneous secretions was measured relative to the reference genes EF1-α and RPL8 using quantitative RT-PCR. Corticosteroid treatment was associated with a significant increase in brevinin-1P gene expression, which was most notable at 24-40 days of corticosteroid administration. Ranatuerin-2P expression followed a similar but non-significant trend. CONCLUSION: This treatment protocol, including corticosteroid-administration and frequent norepinephrine-induced secretion, increased AMP gene expression in the skin of L. pipiens under these experimental conditions. The findings do not support the hypothesis that environmental stress predisposes frogs to chytridiomycosis by causing glucocorticoid-induced suppression of antimicrobial peptide defences.

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris.

The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS)1, L2: (GGGGS)2, and L3:(GGGGS)3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.

Pesticide concentrations in frog tissue and wetland habitats in a landscape dominated by agriculture.

Habitat loss and exposure to pesticides are likely primary factors contributing to amphibian decline in agricultural landscapes. Conservation efforts have attempted to restore wetlands lost through landscape modifications to reduce contaminant loads in surface waters and providing quality habitat to wildlife. The benefits of this increased wetland area, perhaps especially for amphibians, may be negated if habitat quality is insufficient to support persistent populations. We examined the presence of pesticides and nutrients in water and sediment as indicators of habitat quality and assessed the bioaccumulation of pesticides in the tissue of two native amphibian species Pseudacris maculata (chorus frogs) and Lithobates pipiens (leopard frogs) at six wetlands (3 restored and 3 reference) in Iowa, USA. Restored wetlands are positioned on the landscape to receive subsurface tile drainage water while reference wetlands receive water from overland run-off and shallow groundwater sources. Concentrations of the pesticides frequently detected in water and sediment samples were not different between wetland types. The median concentration of atrazine in surface water was 0.2 µg/L. Reproductive abnormalities in leopard frogs have been observed in other studies at these concentrations. Nutrient concentrations were higher in the restored wetlands but lower than concentrations thought lethal to frogs. Complex mixtures of pesticides including up to 8 fungicides, some previously unreported in tissue, were detected with concentrations ranging from 0.08 to 1,500 µg/kg wet weight. No significant differences in pesticide concentrations were observed between species, although concentrations tended to be higher in leopard frogs compared to chorus frogs, possibly because of differences in life histories. Our results provide information on habitat quality in restored wetlands that will assist state and federal agencies, landowners, and resource managers in identifying and implementing conservation and management actions for these and similar wetlands in agriculturally dominated landscapes.

Vitellogenin of the northern leopard frog (Rana pipiens): development of an ELISA assay and evaluation of induction after immersion in xenobiotic estrogens.

An immunoassay for leopard frog (Rana pipiens) vitellogenin was developed for studying endocrine disruption. Male frogs were injected with estradiol-17ß to stimulate vitellogenin for purification. SDS-PAGE revealed high amounts of a 170-180 kDa protein, which was confirmed to be vitellogenin by Western blotting. Vitellogenin was purified by DEAE chromatography and used to generate a polyclonal antibody. A competitive ELISA was developed for leopard frog vitellogenin with a detection limit of 6.0 ng mL(-1) and a working range of 20-1000 ng mL(-1). The intra-assay coefficient of variation averaged 5.47% for control sera and 9.71% for estrogen-treated sera. The inter-assay coefficient of variation averaged 8.21% for control sera and 9.93% for estrogen-treated sera. Recovery of purified vitellogenin averaged 95.2%. Vitellogenin was measured in male frogs immersed in the estrogenic compound diethylstilbestrol (DES) for various times and doses. Serum vitellogenin was detected within five days after immersion in 1.0 mg L(-1) DES and levels continued to increase through 20 d. In a 20-day dose-response experiment, serum vitellogenin was detected in frogs immersed in 0.01 mg L(-1) DES and vitellogenin concentration increased with dose. Immersion of frogs in one of several xenobiotic estrogens (nonylphenol, octylphenol, bisphenol-A) for 20 d did not increase vitellogenin for any treatment, suggesting that this frog may be less sensitive than fish to endocrine disruptors. Vitellogenin induction in R.pipiens may be a useful amphibian model system for field studies of endocrine disruption, due to its broad geographic range.

Consequences of proline-to-alanine substitutions for the stability and refolding of onconase.

Peptidyl-prolyl isomerization reactions can make for rate-limiting steps in protein folding due to their high activation energy. Onconase, an unusually stable ribonuclease A homologue from the Northern leopard frog, contains four trans proline residues in its native state. During the refolding from its guanidine hydrochloride unfolded state, which includes the formation of a folding intermediate, the slowest of the three phases has earlier been attributed to a cis-to-trans peptidyl-prolyl isomerization reaction. We thus substituted all four proline residues individually by alanine and investigated the effect of the amino acid substitutions on the folding and stability of the onconase variants. All onconase variants proved to adopt a tertiary structure comparable with that of the wild-type protein. Although the slow phase was not eliminated for any of the variants, the P43A substitution resulted in an increase in the rate constant of the fast folding phase, i.e. a faster formation of the folding intermediate. This variant also exhibits a significant increase in thermodynamic stability. As residue 43 belongs to those residues that are protected from hydrogen exchange with the solvent in the folding intermediate, the increase in the rate constant and stability of the P43A variant emphasizes the importance of the intermediate for the folding of onconase.

Toxicokinetics of polybrominated diphenyl ethers across life stages in the northern leopard frog (Lithobates pipiens).

Polybrominated diphenyl ethers (PBDEs), a class of flame retardants, are bioaccumulative toxins that can biomagnify in food webs. However, little is known about the toxicokinetics of total and congener-specific BDEs in lower vertebrates. The authors exposed northern leopard frog (Lithobates (Rana) pipiens) tadpoles to diets containing DE-71 (a pentabromodiphenyl ether mixture (0 ng/g as control, 71.4 ng/g, and 634 DE-71 ng/g wet mass)) for 50 d, followed by a period of depuration during which they were fed only undosed (control) food. After 28 d, tadpoles eliminated over 94% of the ΣPBDEs from their tissues (t½ = 5.9 ± 1.9 d) with no significant differences in elimination rates for the predominant congeners. Elimination of BDE-99 was independent of dose, indicating first-order kinetics. It did not fit a biexponential model significantly better than a monoexponential model, indicating single-compartment elimination. To compare developmental life-stage kinetics following larval exposure, the authors collected individuals at the beginning and end of metamorphosis and at 70 d postmetamorphosis. During metamorphosis, total-body residues per individual did not significantly change, implying little to no elimination. After 70 d, juvenile frogs eliminated 89.7% of the ΣPBDEs from their tissues, and BDE-47 was eliminated at a faster rate (t½ = 17.3 d) than BDE-99 and BDE-100 (t½ = 63.0 d and 69.3 d, respectively). Because the kinetics of PBDEs in L. pipiens differed among life stages, developmental life stage-especially for species that undergo metamorphosis-should be considered when determining the toxicity of persistent organic pollutants.

Evidence for water-permeable channels in auditory hair cells in the leopard frog.

Auditory hair cells in the amphibian papilla (APHCs) of the leopard frog, Rana pipiens pipiens, have a significantly higher permeability to water than that observed in mammalian hair cells. The insensitivity of water permeability in frog hair cells to extracellular mercury suggests that an amphibian homologue of the water channel aquaporin-4 (AQP4) may mediate water transport in these cells. Using immunocytochemistry, we show that an AQP4-like protein is found in APHCs. Rabbit anti-AQP4 antibody was used in multiple-immunohistochemical staining experiments along with AP hair cell and hair bundle markers in leopard frog and mouse tissue. AQP4 immunoreactivity was found in the basal and apical poles of the APHCs and shows uniform immunoreactivity. This study provides the first identification and localization of an AQP4-like protein in the amphibian inner ear. We also report a more direct measure of hyperosmotically-induced volume changes in APHCs that confirms previous findings. The presence of water channels in anuran APHCs constitutes a novel physiological difference between amphibian and mammalian hair cell structure and function.

Physiological effects and tissue residues from exposure of leopard frogs to commercial naphthenic acids.

Naphthenic acids (NAs) have been cited as one of the main causes of the toxicity related to oil sands process-affected materials and have recently been measured in biological tissues (fish). However, adverse effects have not been a consistent finding in toxicology studies on vertebrates. This study set out to determine two factors: 1) whether exposure to commercial NAs (Refined Merichem) resulted in detectable tissue residues in native amphibians (northern leopard frogs, Lithobates pipiens), and 2) whether such exposure would produce clinical or subclinical toxicity. Frogs were kept in NA solutions (0, 20, or 40 mg/L) under saline conditions comparable to that on reclaimed wetlands in the Athabasca oil sands for 28 days. These exposures resulted in proportional NA concentrations in muscle tissue of the frogs, estimated by gas chromatography-mass spectrometry analyses. Detailed studies determined if the increasing concentrations of NAs, and subsequently increased tissue NA levels, caused a proportional compromise in the health of the experimental animals. Physiological investigations included innate immune function, thyroid hormone levels, and hepatic detoxification enzyme induction, none of which differed in response to increased exposures or tissue concentrations of NAs. Body mass did increase in both the salt- and NA-exposed animals, likely related to osmotic pressure and uptake of water through the skin. Our results demonstrate that commercial NAs are absorbed and deposited in muscle tissue, yet they show few negative physiological or toxicological effects on the frogs.

Three-state thermal unfolding of onconase.

Onconase is a member of the ribonuclease A superfamily currently in phase IIIb clinical trials as a treatment for malign mesothelioma due to its cytotoxic activity selective against tumor-cells. In this work, we have studied the equilibrium thermal unfolding of onconase using a combination of several structural and biophysical techniques. Our results indicate that at least one significantly populated intermediate, which implies the exposure of hydrophobic surface and significant changes in the environment around Trp3, occurs during the equilibrium unfolding process of this protein. The intermediate begins to populate at about 30° below the global unfolding temperature, reaching a maximum population of nearly 60%, 10° below the global unfolding temperature.

The fungicide chlorothalonil is nonlinearly associated with corticosterone levels, immunity, and mortality in amphibians.

BACKGROUND: Contaminants have been implicated in declines of amphibians, a taxon with vital systems similar to those of humans. However, many chemicals have not been thoroughly tested on amphibians or do not directly kill them. OBJECTIVE: Our goal in this study was to quantify amphibian responses to chlorothalonil, the most commonly used synthetic fungicide in the United States. METHODS: We reared Rana sphenocephala (southern leopard frog) and Osteopilus septentrionalis (Cuban treefrog) in outdoor mesocosms with or without 1 time (1×) and 2 times (2×) the expected environmental concentration (EEC) of chlorothalonil (~ 164 µg/L). We also conducted two dose-response experiments on O. septentrionalis, Hyla squirella (squirrel treefrog), Hyla cinerea (green treefrog), and R. sphenocephala and evaluated the effects of chlorothalonil on the stress hormone corticosterone. RESULTS: For both species in the mesocosm experiment, the 1× and 2× EEC treatments were associated with > 87% and 100% mortality, respectively. In the laboratory experiments, the approximate EEC caused 100% mortality of all species within 24 hr; 82 µg/L killed 100% of R. sphenocephala, and 0.0164 µg/L caused significant tadpole mortality of R. sphenocephala and H. cinerea. Three species showed a nonmonotonic dose response, with low and high concentrations causing significantly greater mortality than did intermediate concentrations or control treatments. For O. septentrionalis, corticosterone exhibited a similar nonmonotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities (< 16.4 µg/L). CONCLUSIONS: Chlorothalonil killed nearly every amphibian at the approximate EEC; at concentrations to which humans are commonly exposed, it increased mortality and was associated with elevated corticosterone levels and changes in immune cells. Future studies should directly quantify the effects of chlorothalonil on amphibian populations and human health.

Carbaryl concentration gradients in realistic environments and their influence on our understanding of the tadpole food web.

Although exposure to pesticides has been correlated with amphibian declines, the mechanism of their role remains enigmatic. Declines have been associated with sublethal exposure, but few outdoor studies have evaluated impacts of low pesticide concentrations. Understanding the effects of a range of pesticide concentrations on amphibians in outdoor mesocosms provides a framework for both direct and indirect effects of exposure. Indirect effects are challenging to glean from lab studies, which typically lack a food web. Our design tested direct and indirect effects of exposure to the insecticide carbaryl on the American toad (Bufo americanus) and the northern leopard frog (Rana pipiens) tadpole survival and growth. We evaluated the effects of five concentrations (2.0, 0.2, 0.02, 0.002, and 0 mg carbaryl/l). Specifically, we predicted a threshold effect in which carbaryl concentrations great enough to reduce zooplankton abundance would have negative indirect effects on tadpoles, but the degree of these effects would not be concentration-specific. Similarly, we predicted that lower carbaryl concentrations (where zooplankton abundance was not reduced) would not differ in effect from controls. We did not observe a threshold effect or any negative effect on tadpoles. The highest carbaryl concentration expedited the time to metamorphosis in the northern leopard frogs by ~4 days. The toads were unaffected by any concentration of carbaryl. Despite significant reductions in zooplankton abundance and increases in phytoplankton abundance, periphyton abundance was unaffected by carbaryl. Taken together, these results suggest that despite evidence from studies using single concentrations of pesticides, trophic cascade models do not sufficiently explain sublethal effects on larval amphibians.

Mechanisms of chloride uptake in frog olfactory receptor neurons.

Odorant stimulation of olfactory receptor neurons (ORNs) leads to the activation of a Ca(2+) permeable cyclic nucleotide-gated (CNG) channel followed by opening of an excitatory Ca(2+)-activated Cl(-) channel, which carries about 70% of the odorant-induced receptor current. This requires ORNs to have a [Cl(-)](i) above the electrochemical equilibrium to render this anionic current excitatory. In mammalian ORNs, the Na(+)-K(+)-2Cl(-) co-transporter 1 (NKCC1) has been characterized as the principal mechanism by which these neurons actively accumulate Cl(-). To determine if NKCC activity is needed in amphibian olfactory transduction, and to characterize its cellular location, we used the suction pipette technique to record from Rana pipiens ORNs. Application of bumetanide, an NKCC blocker, produced a 50% decrease of the odorant-induced current. Similar effects were observed when [Cl(-)](i) was decreased by bathing ORNs in low Cl(-) solution. Both manipulations reduced only the Cl(-) component of the current. Application of bumetanide only to the ORN cell body and not to the cilia decreased the current by again about 50%. The results show that NKCC is required for amphibian olfactory transduction, and suggest that the co-transporter is located basolaterally at the cell body although its presence at the cilia could not be discarded.

Preexposure to ultraviolet B radiation and 4-tert-octylphenol affects the response of Rana pipiens tadpoles to 3,5,3'-triiodothyronine.

Exposure to multiple environmental stressors is negatively impacting the health of amphibians worldwide. Increased exposure to ultraviolet B radiation (UVBR) and chemical pollutants may affect amphibian populations by disrupting metamorphosis; however, the actual mechanisms by which these stressors affect development remain unknown. Because amphibian metamorphosis is controlled by thyroid hormones (TH), changes in developmental rates by environmental stress suggest a disruption of the thyroid system. Tadpoles were chronically exposed to environmental levels of UVBR (average of 0.15 W/m2) and 4-tert-octylphenol (OP; 10 nM), alone and combined, prior to being challenged to exogenous TH triiodothyronine (T3; 5 or 50 nM). This experimental approach was taken to determine whether exposure to these stressors affects the ability of T3 to elicit specific molecular and morphological responses. Exposure to OP increased mRNA levels of thyroid receptors (TRs) alpha and beta, deiodinase type 2 (D2), and corticotropin releasing hormone in the brain and of D2 in the tail of tadpoles. 4-tert-octylphenol also enhanced T3-induced expression of D2 in the brain. The combination of UVBR and OP affected the expression of TR alpha in the brain and the responses of TR alpha and beta genes to T3 in the tail, demonstrating the importance of considering the effects of multiple stressors on amphibians. Tadpoles exposed to UVBR were developmentally delayed and exhibited slowed tail resorption and accelerated hindlimb development following exposure to T3. Together, these findings indicate that UVBR alters the rate of development and TH-dependent morphological changes at metamorphosis, and that exposure to UVBR and/or OP disrupts the expression of genes important for development and the biological action of T3 in peripheral tissues. Our group is the first to demonstrate that environmental levels of UVBR and/or OP can affect the thyroid system of amphibians.

Assessment of thyroid system disruption in Rana pipiens tadpoles chronically exposed to UVB radiation and 4-tert-octylphenol.

Many studies have considered recent increases in ultraviolet B radiation (UVBR) and endocrine disrupting chemicals polluting the environment as possible contributing factors to the reduction in amphibian populations. It has been demonstrated that exposure of amphibians to estrogenic chemicals or UVBR can affect the timing of larval development and metamorphosis. However, amphibians in the wild are exposed to multiple environmental stressors simultaneously. Therefore, our study examines the effects of UVBR and the estrogenic chemical 4-tert-octylphenol (OP), alone and in combination, on the thyroid system of Rana pipiens tadpoles, which is the main regulator of amphibian metamorphosis. Results demonstrate that thyroid gland histomorphology measurements in Gosner stage 31 tadpoles continuously exposed to UVBR (0.21W/m(2)) were not different than those measured in animals from the control group. In a separate experiment, tadpoles exposed to environmentally relevant levels of UVBR (0.22W/m(2)) and/or OP (0.01nM or 10nM) exhibited significantly delayed development starting from Gosner stage 29, given that fewer tadpoles developed past stage 29 in these groups. In addition, significantly fewer UVBR-treated tadpoles developed past stage 34 and metamorphosed. Samples were collected from stages 29 and 34 tadpoles for gene expression analysis in tail tissue and measurements of T3 (triiodothyronine) whole body levels (minus tail). UVBR and/or OP exposure did not affect T3 levels in stages 29 and 34 tadpoles. However, a decrease in deiodinase type 2 (D2) or increase in deiodinase type 3 (D3) mRNA levels was observed in groups of tadpoles with slowed developmental rates at those developmental stages. Given that D2 activates and D3 inactivates thyroid hormones (TH), UVBR/OP mediated disruptions in development are likely caused by dysfunctions in the localized metabolism of THs through alterations in the expression of these enzymes in peripheral tissues. This is the first study to our knowledge reporting a potential thyroid-based mechanism of action for the developmental delays in amphibians exposed to UVBR and/or OP.

Onconase cytotoxicity relies on the distribution of its positive charge.

Onconase (ONC) is a member of the ribonuclease A superfamily that is toxic to cancer cells in vitro and in vivo. ONC is now in Phase IIIb clinical trials for the treatment of malignant mesothelioma. Internalization of ONC to the cytosol of cancer cells is essential for its cytotoxic activity, despite the apparent absence of a cell-surface receptor protein. Endocytosis and cytotoxicity do, however, appear to correlate with the net positive charge of ribonucleases. To dissect the contribution made by the endogenous arginine and lysine residues of ONC to its cytotoxicity, 22 variants were created in which cationic residues were replaced with alanine. Variants with the same net charge (+2 to +5) as well as equivalent catalytic activity and conformational stability were found to exhibit large (> 10-fold) differences in toxicity for the cells of a human leukemia line. In addition, a more cationic ONC variant could be either much more or much less cytotoxic than a less cationic variant, again depending on the distribution of its cationic residues. The endocytosis of variants with widely divergent cytotoxic activity was quantified by flow cytometry using a small-molecule fluorogenic label, and was found to vary by twofold or less. This small difference in endocytosis did not account for the large difference in cytotoxicity, implicating the distribution of cationic residues as being critical for lipid-bilayer translocation subsequent to endocytosis. This finding has fundamental implications for understanding the interaction of ribonucleases and other proteins with mammalian cells.