In skeletal muscle, excitation may cause loss of K+, increased extracellular K+ ([K+]o), intracellular Na+ ([Na+]i), and depolarization. Since these events interfere with excitability, the processes of excitation can be self-limiting. During work, therefore, the impending loss of excitability has to be counterbalanced by prompt restoration of Na+-K+ gradients. Since this is the major function of the Na+-K+ pumps, it is crucial that their activity and capacity are adequate. This is achieved in two ways: 1) by acute activation of the Na+-K+ pumps and 2) by long-term regulation of Na+-K+ pump content or capacity. 1) Depending on frequency of stimulation, excitation may activate up to all of the Na+-K+ pumps available within 10 s, causing up to 22-fold increase in Na+ efflux. Activation of the Na+-K+ pumps by hormones is slower and less pronounced. When muscles are inhibited by high [K+]o or low [Na+]o, acute hormone- or excitation-induced activation of the Na+-K+ pumps can restore excitability and contractile force in 10-20 min. Conversely, inhibition of the Na+-K+ pumps by ouabain leads to progressive loss of contractility and endurance. 2) Na+-K+ pump content is upregulated by training, thyroid hormones, insulin, glucocorticoids, and K+ overload. Downregulation is seen during immobilization, K+ deficiency, hypoxia, heart failure, hypothyroidism, starvation, diabetes, alcoholism, myotonic dystrophy, and McArdle disease. Reduced Na+-K+ pump content leads to loss of contractility and endurance, possibly contributing to the fatigue associated with several of these conditions. Increasing excitation-induced Na+ influx by augmenting the open-time or the content of Na+ channels reduces contractile endurance. Excitability and contractility depend on the ratio between passive Na+-K+ leaks and Na+-K+ pump activity, the passive leaks often playing a dominant role. The Na+-K+ pump is a central target for regulation of Na+-K+ distribution and excitability, essential for second-to-second ongoing maintenance of excitability during work.

Na+-K+ Pump Regulation and Skeletal Muscle Contractility

The herbicide atrazine is one of the most commonly applied pesticides in the world. As a result, atrazine is the most commonly detected pesticide contaminant of ground, surface, and drinking water. Atrazine is also a potent endocrine disruptor that is active at low, ecologically relevant concentrations. Previous studies showed that atrazine adversely affects amphibian larval development. The present study demonstrates the reproductive consequences of atrazine exposure in adult amphibians. Atrazine-exposed males were both demasculinized (chemically castrated) and completely feminized as adults. Ten percent of the exposed genetic males developed into functional females that copulated with unexposed males and produced viable eggs. Atrazine-exposed males suffered from depressed testosterone, decreased breeding gland size, demasculinized/feminized laryngeal development, suppressed mating behavior, reduced spermatogenesis, and decreased fertility. These data are consistent with effects of atrazine observed in other vertebrate classes. The present findings exemplify the role that atrazine and other endocrine-disrupting pesticides likely play in global amphibian declines.

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Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis)

Greater than 70% of the world's amphibian species are in decline. We propose that there is probably not a single cause for global amphibian declines and present a three-tiered hierarchical approach that addresses interactions among and between ultimate and proximate factors that contribute to amphibian declines. There are two immediate (proximate) causes of amphibian declines: death and decreased recruitment (reproductive failure). Although much attention has focused on death, few studies have addressed factors that contribute to declines as a result of failed recruitment. Further, a great deal of attention has focused on the role of pathogens in inducing diseases that cause death, but we suggest that pathogen success is profoundly affected by four other ultimate factors: atmospheric change, environmental pollutants, habitat modification and invasive species. Environmental pollutants arise as likely important factors in amphibian declines because they have realized potential to affect recruitment. Further, many studies have documented immunosuppressive effects of pesticides, suggesting a role for environmental contaminants in increased pathogen virulence and disease rates. Increased attention to recruitment and ultimate factors that interact with pathogens is important in addressing this global crisis.

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The cause of global amphibian declines: a developmental endocrinologist's perspective

Glyphosate [N-(phosphonomethyl) glycine] is a broad spectrum, post emergent herbicide and is among the most widely used agricultural chemicals globally. Initially developed to control the growth of weed species in agriculture, this herbicide also plays an important role in both modern silviculture and domestic weed control. The creation of glyphosate tolerant crop species has significantly increased the demand and use of this herbicide and has also increased the risk of exposure to non-target species. Commercially available glyphosate-based herbicides are comprised of multiple, often proprietary, constituents, each with a unique level of toxicity. Surfactants used to increase herbicide efficacy have been identified in some studies as the chemicals responsible for toxicity of glyphosate-based herbicides to non-target species, yet they are often difficult to chemically identify. Most glyphosate-based herbicides are not approved for use in the aquatic environment; however, measurable quantities of the active ingredient and surfactants are detected in surface waters, giving them the potential to alter the physiology of aquatic organisms. Acute toxicity is highly species dependant across all taxa, with toxicity depending on the timing, magnitude, and route of exposure. The toxicity of glyphosate to amphibians has been a major focus of recent research, which has suggested increased sensitivity compared with other vertebrates due to their life history traits and reliance on both the aquatic and terrestrial environments. This review is designed to update previous reviews of glyphosate-based herbicide toxicity, with a focus on recent studies of the aquatic toxicity of this class of chemicals.

Impact of glyphosate and glyphosate‐based herbicides on the freshwater environment

Amphibians, a class of animals in global decline, are present in agricultural landscapes characterized by agrochemical inputs. Effects of pesticides on terrestrial life stages of amphibians such as juvenile and adult frogs, toads and newts are little understood and a specific risk assessment for pesticide exposure, mandatory for other vertebrate groups, is currently not conducted. We studied the effects of seven pesticide products on juvenile European common frogs (Rana temporaria) in an agricultural overspray scenario. Mortality ranged from 100% after one hour to 40% after seven days at the recommended label rate of currently registered products. The demonstrated toxicity is alarming and a large-scale negative effect of terrestrial pesticide exposure on amphibian populations seems likely. Terrestrial pesticide exposure might be underestimated as a driver of their decline calling for more attention in conservation efforts and the risk assessment procedures in place do not protect this vanishing animal group.

/pdf/terrestrial-pesticide-exposure-of-amphibians-an-523va3ewmg.pdf

Terrestrial pesticide exposure of amphibians: An underestimated cause of global decline?

Dramatic declines in amphibian populations have been described all over the world since the 1980s. The evidence that the sensitivity to environmental threats is greater in amphibians than in mammals has been generally linked to the observation that amphibians are characterized by a rather permeable skin. Nevertheless, a numerical comparison of data of percutaneous (through the skin) passage between amphibians and mammals is lacking. Therefore, in this investigation we have measured the percutaneous passage of two test molecules (mannitol and antipyrine) and three heavily used herbicides (atrazine, paraquat and glyphosate) in the skin of the frog Rana esculenta (amphibians) and of the pig ear (mammals), by using the same experimental protocol and a simple apparatus which minimizes the edge effect, occurring when the tissue is clamped in the usually used experimental device.

The percutaneous passage (P) of each substance is much greater in frog than in pig. LogP is linearly related to logKow (logarithm of the octanol-water partition coefficient). The measured P value of atrazine was about 134 times larger than that of glyphosate in frog skin, but only 12 times in pig ear skin. The FoD value (Pfrog/Ppig) was 302 for atrazine, 120 for antipyrine, 66 for mannitol, 29 for paraquat, and 26 for glyphosate.

The differences in structure and composition of the skin between amphibians and mammals are discussed.

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Why amphibians are more sensitive than mammals to xenobiotics

The presence of atrazine in agricultural sites has been linked to the decline in amphibian populations. The efforts of the scientific community generally are directed toward investigating the long-term effect of atrazine on complex functions (reproduction or respiration), but in the present study, we investigated the short-term effect on the short-circuit current (I(sc)), a quantitative measure of the ion transport operated by frog (Rana esculenta) skin. Treatment with 5 microM atrazine (1.08 mg/L) does not affect the transepithelial outfluxes of [14C]mannitol or [14C]urea; therefore, atrazine does not damage the barrier properties of frog skin. Atrazine causes a dose-dependent increase in the short-circuit current, with a minimum of 4.64 +/- 0.76 microA/cm2 (11.05% +/- 1.22%) and a maximum of 12.7 +/- 0.7 microA/cm2 (35% +/- 2.4%) measured at 10 nM and 5 microM, respectively. An increase in Isc also is caused by 5 microM ametryne, prometryn, simazine, terbuthylazine, or terbutryn (other atrazine derivatives). In particular, atrazine increases the transepithelial 22Na+ influx without affecting the outflux. Finally, stimulation of Isc by atrazine is suppressed by SQ 22536, H89, U73122, 2-aminoethoxydiphenyl borate, and W7 (blockers of adenylate cyclase, protein kinase A, phospholipase C, intracellular Ca2+ increase, and calmodulin, respectively), whereas indomethacin and calphostin C (inhibitors of cyclooxygenase and protein kinase C, respectively) have no effect.

Atrazine increases the sodium absorption in frog (Rana esculenta) skin.

Capsaicin at low concentrations increases the short circuit current (SCC) across frog skin. Simultaneous measurements of both transepithelial fluxes of 22Na or 36Cl demonstrate that the SCC increase is due to stimulation of sodium active absorption. Capsaicin acts through the liberation of several peptides; thus these peptides were tested on the SCC across frog skin. Those more active are, in order of potency: Cyclic Calcitonin Gene Related Peptide (CGRP), Kassinin and Eledoisin, Substance P (SP) and Neurokinin A. Neurokinin B and Vasoactive Intestinal Peptide (VIP) have no effect. Also the actions of SP and CGRP are due mainly to stimulation of Na+ active absorption. A strict parallelism regarding the sensitivity to inhibitors (Naproxen, SQ22536 and CP96345) between SP, CGRP and Capsaicin strengthens the hypothesis that SP and CGRP are liberated by Capsaicin in this tissue.

Action of capsaicin and related peptides on the ionic transport across the skin of Rana esculenta.

Pyrethroids are grouped into two classes (types I and II) because of the absence or presence of an α-cyano substituent and the production of a different intoxication syndrome in rodents. In this study, we investigated the effect of pyrethroids on the ion transport across frog skin (Rana esculenta). The short-circuit current value (estimate of ion transport) was increased by each of the eight pyrethroids tested, with the following order of potency: lambda-cyhalothrin > deltamethrin > alpha-cypermethrin = beta-cyfluthrin > bioallethrin > permethrin > bioresmethrin > phenothrin. The first four compounds are type II pyrethroids. Therefore, ion transport is stimulated more by type II pyrethroids than by type I. Experiments performed in the presence of amiloride support the conclusion that pyrethroids mainly increase Na + absorption and to a lesser extent Cl secretion. In these experiments, no systematic difference between type I and II pyrethroids was found. Finally, the stimulation by pyrethroids was inhibited by indomethacin and W7 (inhibitors of cyclooxygenases and the Ca 2+ /calmodulin system, respectively). These observations suggest that pyrethroids do not directly affect the epithelial Na + channel (ENaC) but indirectly influence an intracellular event involved in ENaC modulation and linked to the Ca 2+ signaling cascade.

Pyrethroid stimulation of ion transport across frog skin.

We have measured, in the edible frog (Pelophylax kl. esculentus), the effect of two fungicides (8-hydroxyquinoline and captan), and four herbicides (DCMU, glyphosate, paraquat, and propachlor) on the short-circuit current, whose value gives an estimate of the net ion transport taking place across isolated skin. Glyphosate and paraquat treatment produced a modest increase in short-circuit current, corresponding to 2.6±0.7 and 4.6±0.8 μA·cm−2, whereas the other substances had a more sustained effect, ranging from 9.1±0.6 (propachlor) to 14.8±0.9 μA·cm−2 (captan), which is mainly attributable to an increase in the Na+ absorption, and, to a lesser extent, Cl− secretion. The increase in short-circuit current after pesticide treatment, was partially abolished by AF12198, indomethacin, SC58125, SQ 22536, and W7; these results suggest that pesticides, independently from their chemical structure, induce the release of interleukin-1, which triggers the activity of cyclooxygenase-2, whose products, via a concentrati...

Vito Bellantuono

Papers

Why amphibians are more sensitive than mammals to xenobiotics

Atrazine increases the sodium absorption in frog (Rana esculenta) skin.

Action of capsaicin and related peptides on the ionic transport across the skin of Rana esculenta.

Pyrethroid stimulation of ion transport across frog skin.

Pesticides alter ion transport across frog (Pelophylax kl. esculentus) skin