Multi-species Aquatic Toxicity Assessment of 1-Methyl-3-Nitroguanidine (MeNQ).

The Army is replacing traditional munitions with insensitive munitions (IM) resistant to accidental detonation. The aquatic toxicity of 1-methyl-3-nitroguanidine (MeNQ), which is being assessed for potential use in IM formulations, remains largely untested. The present study fills a number of critical data gaps for MeNQ aquatic toxicity by evaluating effects across two vertebrate and five invertebrate species. Specifically, responses in larval Pimephales promelas, Rana pipiens tadpoles, Chironomus dilutus, Lumbriculus variegatus, Hydra littoralis, Hyalella azteca, and Daphnia pulex were assessed in MeNQ exposures across various acute, subchronic, and chronic bioassays. Overall, survival was unaffected in most of the MeNQ exposures where significant lethal effects were only observed in D. pulex, H. littoralis, and C. dilutus and only at concentrations ≥ 2186 mg/L. Significant sublethal effects on growth were observed for C. dilutus at 903 mg/L and H. azteca at 1098 mg/L in 10-d assays. Significantly decreased reproduction was observed at 2775 mg/L for H. azteca in a chronic 35-d assay and at 174 mg/L for D. pulex in the 11-d three-brood assay representing a sublethal effect one order of magnitude more sensitive than the effective lethal concentration for D. pulex (2987 mg/L). Degradation of MeNQ in ultraviolet light (UV) greatly increased toxicity to D. pulex. Specifically, exposure to a MeNQ solution that was completely UV-degraded prior to D. pulex exposures resulted in an 11-d LC50 of 6.1 mg/L and a 50% reduction in reproduction at 3.125 mg/L, based on the original MeNQ parent-compound concentrations.

Assessment of sublethal ecotoxicity of solvents on larvae of a model native amphibian (Lithobates pipiens).

Carrier solvents are used frequently in toxicity testing to assist hydrophobic chemicals into solution, but such solvents may have toxic effects on test subjects. Amphibians are model organisms in toxicity studies; however, little is known about the direct effects of solvents on native amphibians. Following modifications to standardized guidelines for native species, we used acute 96-hour exposures to assess the direct effects of three common solvents on survival, differences in morphology and occurrence of abnormalities of northern leopard frog larvae (Lithobates pipiens). The solvents, dimethyl sulfoxide (DMSO), ethanol (ETOH) and acetone (ACE) were used at nominal concentrations ranging from 1 to 100 µL/L. We also conducted a 30-day exposure to assess the direct chronic effects of DMSO at 1 and 5 µL/L, on larval growth, development and sex differentiation, but found no effects. Acute exposure to solvents also had no effect on the survival of larvae, but we found significant abnormalities in tadpoles acutely exposed to 100 µL/L ACE. Acute exposure to DMSO and ETOH had further concentration-dependent effects on larval morphological traits. Our study suggests that DMSO and ETOH at ≤20 µL/L may be used as solvents in amphibian ecotoxicological studies, but ACE should be limited to ≤50 µL/L in ecotoxicity studies and perhaps much less (≤10 µL/L) in studies with other amphibians, based on a review of existing literature. We emphasize pilot studies when using solvents on acute and chronic ecotoxicity tests, using native amphibians.

Assessment of Sublethal Effects of Neonicotinoid Insecticides on the Life-History Traits of 2 Frog Species.

Neonicotinoid insecticides are used extensively in agriculture and, as a consequence, are now detectable in nearby aquatic environments. Few studies have evaluated the effects of neonicotinoids on amphibians in these aquatic environments. In the present study, we examined the effects of 2 commercial formulations of neonicotinoids (active ingredients clothianidin and thiamethoxam) on survival and life-history traits of wood frogs (Lithobates sylvaticus) and northern leopard frogs (Lithobates pipiens). We used artificial pond mesocosms to assess the effects of these neonicotinoids, at nominal concentrations of 2.5 and 250 µg/L, on amphibian larval development through metamorphosis. We found no differences between controls and neonicotinoid exposure for any of the endpoints assessed for either wood frogs or leopard frogs. The present study suggests that concentrations meeting or exceeding observed levels of clothianidin and thiamethoxam in surface waters will not directly affect metamorphosis in 2 amphibians. Environ Toxicol Chem 2019;38:1967-1977. © 2019 SETAC.

Functional optical coherence tomography enables in vivo physiological assessment of retinal rod and cone photoreceptors.

Transient intrinsic optical signal (IOS) changes have been observed in retinal photoreceptors, suggesting a unique biomarker for eye disease detection. However, clinical deployment of IOS imaging is challenging due to unclear IOS sources and limited signal-to-noise ratios (SNRs). Here, by developing high spatiotemporal resolution optical coherence tomography (OCT) and applying an adaptive algorithm for IOS processing, we were able to record robust IOSs from single-pass measurements. Transient IOSs, which might reflect an early stage of light phototransduction, are consistently observed in the photoreceptor outer segment almost immediately (<4 ms) after retinal stimulation. Comparative studies of dark- and light-adapted retinas have demonstrated the feasibility of functional OCT mapping of rod and cone photoreceptors, promising a new method for early disease detection and improved treatment of diseases such as age-related macular degeneration (AMD) and other eye diseases that can cause photoreceptor damage.

Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction.

The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium channels and synaptic vesicles, the flux through a single open calcium channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium channels might be required to contribute sufficient calcium ions to trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of individual synaptic vesicles is predominately triggered by calcium ions entering the nerve terminal through the nearest open calcium channel. Furthermore, calcium ion flux through this channel has a low probability of triggering synaptic vesicle fusion (∼6%), even when multiple channels open in a single active zone. These mechanisms work to control the rare triggering of vesicle fusion in the frog neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.

Single-pixel optical fluctuation analysis of calcium channel function in active zones of motor nerve terminals.

We used high-resolution fluorescence imaging and single-pixel optical fluctuation analysis to estimate the opening probability of individual voltage-gated calcium (Ca(2+)) channels during an action potential and the number of such Ca(2+) channels within active zones of frog neuromuscular junctions. Analysis revealed ∼36 Ca(2+) channels within each active zone, similar to the number of docked synaptic vesicles but far less than the total number of transmembrane particles reported based on freeze-fracture analysis (∼200-250). The probability that each channel opened during an action potential was only ∼0.2. These results suggest why each active zone averages only one quantal release event during every other action potential, despite a substantial number of docked vesicles. With sparse Ca(2+) channels and low opening probability, triggering of fusion for each vesicle is primarily controlled by Ca(2+) influx through individual Ca(2+) channels. In contrast, the entire synapse is highly reliable because it contains hundreds of active zones.

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.

Secretory expression of glycosylated and aglycosylated mutein of onconase from Pichia pastoris using different secretion signals and their purification and characterization.

Onconase, an RNAse extracted from embryos of the Northern leopard frog (Rana pipiens), is in a confirmatory phase IIIb clinical trial for the treatment of unresectable malignant mesothelioma. Because the current purification process for onconase is cumbersome and laborious, the development of more efficient and cost-effective alternative sources is imperative. In this study, we assessed the potential of Pichia pastoris as an expression host for the large-scale production of onconase. Because of its specific N-terminal structure, active onconase with a correct N-terminus could not be secreted by an alpha-mating factor (alpha-MF)-prepro secretion signal, and an alpha-MF-pre secretion signal should be used instead. Onconase accumulated to a high concentration (about 300 and 150 mg L(-1) for glycosylated onconase and aglycosylated mutein, respectively) in high cell density fermentation, and was purified to homogeneity with high yields (56% for glycosylated onconase and 67% for aglycosylated mutein) by a simple purification process consisting of cation exchange chromatography and size exclusion chromatography. In vitro activity assays revealed that glycosylation decreased both the RNAse activity and the cytotoxic activity of onconase. The high expression level and subsequent facile purification process make P. pastoris an efficient and cost-effective host for the large-scale production of onconase.

Phorbol esters and adenosine affect the readily releasable neurotransmitter pool by different mechanisms at amphibian motor nerve endings.

Phorbol esters and adenosine have been proposed to interact at common sites downstream of calcium entry at amphibian motor nerve endings. We thus studied the actions and interactions of phorbol esters and adenosine using electrophysiological recording techniques in conjunction with both binomial statistical analysis and high-frequency stimulation at the amphibian neuromuscular junction. To begin this study, we confirmed previous observations that synchronous evoked acetylcholine (ACh) release (reflected as endplate potentials, EPPs) is well described by a simple binomial distribution. We then used binomial analysis to study the effects of the phorbol ester phorbol dibutyrate (PDBu, 100 nM) and adenosine (50 microM) on the binomial parameters n (the number of calcium charged ACh quanta available for release) and p (the average probability of release), where the mean level of evoked ACh release (m) = np. We found that PDBu increased m by increasing the parameter n whilst adenosine reduced m by reducing n; neither agent affected the parameter p. PDBu had no effect on either the potency or efficacy of the inhibition produced by adenosine. Subtle differences between these two agents were revealed by the patterns of EPPs evoked by high-frequency trains of stimuli. Phorbol esters increased ACh release during the early phase of stimulation but not during the subsequent plateau phase. The inhibitory effect of adenosine was maximal at the beginning of the train and was still present with reduced efficacy during the plateau phase. When taken together with previous findings, these present results suggest that phorbol esters increase the immediately available store of synaptic vesicles by increasing the number of primed vesicles whilst adenosine acts at a later stage of the secretory process to decrease the number of calcium-charged primed vesicles.

Intracellular Ca(2+) release as irreversible Markov process.

In striated muscles, intracellular Ca(2+) release is tightly controlled by the membrane voltage sensor. Ca(2+) ions are necessary mediators of this control in cardiac but not in skeletal muscle, where their role is ill-understood. An intrinsic gating oscillation of Ca(2+) release-not involving the voltage sensor-is demonstrated in frog skeletal muscle fibers under voltage clamp. A Markov model of the Ca(2+) release units is shown to reproduce the oscillations, and it is demonstrated that for Markov processes to have oscillatory transients, its transition rates must violate thermodynamic reversibility. Such irreversibility results in permanent cycling of the units through a ring of states, which requires a source of free energy. Inhibition of the oscillation by 20 to 40 mM EGTA or partial depletion of Ca(2+) in the sarcoplasmic reticulum (SR) identifies the SR [Ca(2+)] gradient as the energy source, and indicates a location of the critical Ca(2+)-sensing site at distances greater than 35 nm from the open channel. These results, which are consistent with a recent demonstration of irreversibility in gating of cardiac Ca(2+) sparks, (Wang, S.-Q., L.-S. Song, L. Xu, G. Meissner, E. G. Lakatta, E. Ríos, M. D. Stern, and H. Cheng. 2002. Biophys. J. 83:242-251) exemplify a cell-wide oscillation caused by coupling between ion permeation and channel gating.

Assessment of the risk of solar ultraviolet radiation to amphibians. I. Dose-dependent induction of hindlimb malformations in the northern leopard frog (Rana pipiens).

A number of environmental stressors have been hypothesized as responsible for recent increases in limb malformations in several species of North American amphibians. The purpose of this study was to generate dose-response data suitable for assessing the potential role of solar ultraviolet (UV) radiation in causing limb malformations in a species in which this phenomenon seemingly is particularly prevalent, the northern leopard frog (Rana pipiens). Frogs were exposed from early embryonic stages through complete metamorphosis to varying natural sunlight regimes, including unaltered (100%) sunlight, sunlight subjected to neutral density filtration to achieve relative intensities of 85%, 75%, 65%, 50%, and 25% of unaltered sunlight, and sunlight filtered with glass or acrylamide to attenuate, respectively, the UVB (290-320 nm) and UVB plus UVA (290-380 nm) portions of the spectrum. The experiments were conducted in a controlled setting, with continual monitoring of UVB, UVA, and visible light to support a robust exposure assessment. Full sunlight caused approximately 50% mortality of the frogs during early larval development; no significant treatment-related mortality occurred under any of the other exposure regimes, including 100% sunlight with glass or acrylamide filtration. There was a dose-dependent (p < 0.0001) induction of hindlimb malformations in the frogs, with the percentage of affected animals ranging from about 97% under unaltered sunlight to 0% in the 25% neutral density treatment. Malformations were comprised mostly of missing or truncated digits, and generally were bilateral as well as symmetrical. Filtration of sunlight with either glass or acrylamide both significantly reduced the incidence of malformed limbs. The estimated sunlight dose resulting in a 50% limb malformation rate (ED50) was 63.5%. The limb ED50 values based on measured sunlight intensities corresponded to average daily doses of 4.5 and 100 Wh x m(-2) for UVB and UVA, respectively. Exposure to sunlight also resulted in increased eye malformations in R. pipiens, however, the dose-response relationship for this endpoint was not monotonic. The results of this study, in conjunction with measured or predicted exposure data from natural settings, provide a basis for quantitative prediction of the risk of solar UV radiation to amphibians.

Assessment of the risk of solar ultraviolet radiation to amphibians. II. In situ characterzation of exposure in amphibian habitats.

Ultraviolet B (UVB) radiation has been hypothesized as a potential cause of amphibian population declines and increased incidence of malformations. Realistic studies documenting UV irradiance or dose have rarely been conducted in wetlands used by amphibians. Our data indicates that 99% of UVB is attenuated in the top 5-20 cm of wetlands in our study region (northern Minnesota and Wisconsin). Furthermore, vegetation and other habitat features have substantial impacts on local UVB irradiance levels and dose. UVB attenuation in the water columns of our wetlands is controlled by the specific absorption of dissolved organic carbon (DOC), and consequently, UVB attenuation is best predicted by simple laboratory absorbance measurements such as bulk water color (absorbance at 440 nm) or wavelength-specific absorbance coefficients. Seasonal data indicate thatthe UVB absorption by early and mid-season DOC is higher than that of late summer and fall DOC, suggesting increased protection from UVB during the potentially sensitive stages of amphibian development. In addition to dissolved components, our model indicates that suspended solids play a small role in UVB attenuation in our wetlands but apparently only at high concentrations. Models predicting UV attenuation in wetlands should be used cautiously and should consider temporal variability, given the volatility and dynamic nature of water column characteristics in wetlands. Organism behavior is a critical but poorly understood phenomenon that must be addressed for development of an accurate UV exposure risk model for amphibians.

Evidence for two distinct processes in the final stages of neurotransmitter release as detected by binomial analysis in calcium and strontium solutions.

The statistical parameters underlying acetylcholine (ACh) release were studied using Ca(2+) and Sr(2+) ions to promote ACh secretion. Experiments were performed at frog neuromuscular junctions using electrophysiological recording techniques. Increases in asynchronous ACh release, reflected as the frequency of occurrence of miniature end-plate potentials (MEPP(f)), were evoked by high potassium depolarization in either Ca(2+) or Sr(2+) solutions. Increases in MEPP(f) mediated by Ca(2+) were of very low probability and well-described by a Poisson distribution whilst similar MEPP(f) increases mediated by Sr(2+) were best described as a simple binomial distribution. From the binomial distribution in Sr(2+) solutions, values for the average probability of release (p) and the number of releasable ACh quanta (n) may be determined (whereby mean MEPP(f) = np). In Sr(2+) solutions, values of p were independent of both bin width and of the value of n, suggesting that both n and p were stationary. Calculations of p using the simple binomial distribution in Sr(2+) solutions gave theoretical values for the third moment of the mean which were indistinguishable from the experimental distribution. These results, in conjunction with Monte Carlo simulations of the data, suggest that spatial and temporal variance do not measurably affect the analysis. Synchronous ACh release evoked by nerve impulses (end-plate potentials, EPPs) follow a simple binomial distribution in both Ca(2+) and Sr(2+) solutions. Similar mean levels of synchronous ACh release (m, where m = np) were produced by lower values of p and higher values of n in Ca(2+) as compared to Sr(2+). The statistical analyses suggest the presence of two different Ca(2+)-dependent steps in the final stages of neurotransmitter release. The results are discussed in accordance with (i) statistical models for quantal neurotransmitter release, (ii) the role of Sr(2+) as a partial agonist for evoked ACh release, and (iii) the specific loci that may represent the sites of Ca(2+) and Sr(2+) sensitivity.

Interdomain interactions within ryanodine receptors regulate Ca2+ spark frequency in skeletal muscle.

DP4 is a 36-residue synthetic peptide that corresponds to the Leu(2442)-Pro(2477) region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca(2)+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618-11625). We have investigated the effects of DP4 on local SR Ca(2)+ release events (Ca(2)+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca(2)+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca(2)+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca(2)+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca(2)+ release channel(s) generating the sparks. DP4 also increased [(3)H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca(2)+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca(2)+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg(17) to Cys(17) replacement (DP4mut), which corresponds to the Arg(2458)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg(2)+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg(2)+-free RyR(s), thus promoting channel opening and production of Ca(2)+ sparks.

Toxicity of boron to rainbow trout: a weight-of-the-evidence assessment.

From the large data set available on the toxicity of boron to aquatic organisms, the toxicity of boron to the early life stages of rainbow trout (Oncorhyncus mykiss) is the seminal issue relative to setting water quality criteria and effluent standards. Issues associated with the early life stage studies are the flat concentration-response curve, the low threshold of toxicity, and teratogenic effects observed. Recent laboratory and field studies offer new experimental data that make a weight-of-the-evidence assessment timely. In a re-examination of the effect of boron on the embryo-larval stage in rainbow trout and zebrafish, adverse effects due to boron deficiency are observed which decrease with increasing dose. It was found that low concentrations of boron stimulate embryonic growth in rainbow trout and increase the viability and survival of embryonic zebrafish. As boron concentration is further increased, the dose-response curve becomes flat as homeostatic processes are active; this is followed at higher doses by a new adverse response that increases with increasing dose. As a result, the dose-response relationship is U shaped, consistent with the characteristic shape of an essential micronutrient. Thus, effects originally reported to be toxicity at low exposures rather may be due to boron deficiency. Water analyses in trout hatcheries and field studies in wild trout streams add additional information on the toxicity of boron to trout. Of particular note is a controlled field study carried out in the Firehole River in Yellowstone Park (WY, USA), where trout populations survive and reproduction successfully occurs in natural water containing boron concentrations up to and in some cases greater than 1.0 mg B/L. Teratogenic effects due to boron exposure were not observed in any of these more recent studies.

Network assessment of capillary hydraulic conductivity after abrupt changes in fluid shear stress.

Intact capillaries are composed of endothelial cells that experience continuous fluid shear stress as blood flows. Endothelial cells grown in culture demonstrate changes in barrier function in response to changes in shear stress. Therefore, it was hypothesized that intact capillaries would alter filtration in response to changes in fluid shear stress. Capillaries (n = 25) located in frog mesentery were classified according to flow direction as arteriolar, true, or venular and cannulated at 30 cm H2O to induce an abrupt change in shear stress. Frog and human red blood cells acted as velocity markers before and after cannulation, respectively, for calculating fluid shear stress. Hydraulic conductivity (Lp) was measured at 30 cm H2O following a change in shear stress and ranged from 0.7 to 96. 8 x 10(-7) cm s-1 cm H2O-1. For true and venular capillaries, Lp was related to the magnitude of the shear stress stimulus and accounted for the wide range in absolute values of Lp. Arteriolar capillaries did not respond to the mechanical stimulus. These data indicate that blood flow may modulate filtration in homogeneous subpopulations of capillaries in the capillary bed.

T-lymphocyte and B-lymphocyte dichotomy in anuran amphibians: III. Assessment and identification of inducible killer T-lymphocytes (IKTL) and spontaneous killer T-lymphocytes (SKTL).

We have established the existence of alloreactive inducible killer (IK) T-lymphocytes in Rana pipiens by injecting immunogenic concentrations of allogeneic frog erythrocytes (RBC). Assessment of specific IK activity was determined microscopically, observing effector-target conjugate formation, and spectrophotometrically as released hemoglobin (Hb) from lysed targets (RBC). The presence of spontaneous killer (SK) T-lymphocyte activity was also determined using unimmunized frogs and similar assay conditions. Assays using rabbit anti-frog Thy-1.1 antiserum inhibition, but not E-rosetted T-lymphocyte depletion, confirmed the T-lymphocyte category of both effector cell populations in Rana pipiens. For IK activity, we determined the 1) best priming doses, 2) best effector cell source (peripheral blood), 3) best priming route (intraperitoneal), 4) kinetics of immunity development, and 5) kinetics of lysis. Kinetics of lysis and organ distribution for spontaneous killer cells were also determined. Our results may assist 1) in establishing the evolutionary origin of cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells, and 2) in predicting where the capacity of immuno-surveillance against modified-self appeared in phylogeny. The implications are important for understanding origins of mechanisms of resistance against neoplastic conditions.