Melanophores are pigmented cells that change the distribution of melanosomes, enabling animals to appear lighter or darker for camouflage, thermoregulation, and protection from ultraviolet radiation. A complex series of hormonal and neural mechanisms regulates melanophore pigment distribution, making these dynamic cells a valuable tool to screen toxicants as they rapidly respond to changes in the environment. We found that maltol, a naturally occurring flavor enhancer and fragrance agent, induces melanophore pigment aggregation in a dose-dependent manner in Xenopus laevis tadpoles.To determine if maltol affects camouflage adaptation, we placed tadpoles into maltol baths situated over either a white or a black background. Maltol induced pigment aggregation in a similar dose-dependent pattern regardless of background color. We also tested how maltol treatment compares to melatonin treatment and found that the degree of pigment aggregation induced by maltol is similar to treatment with melatonin but that maltol induces over a much longer time course. Last, maltol had no effect on mRNA expression in the brain of genes that regulate camouflage-related pigment aggregation. The present results suggest that maltol does not exert its effects via the camouflage adaptation mechanism or via melatoninrelated mechanisms. These results are the first to identify a putative toxicological effect of maltol exposure in vivo and rule out several mechanisms by which maltol may exert its effects on pigment aggregation. Environ Toxicol Chem 2020;39:381-395.

Melanophores are pigmented cells that change the distribution of pigmented melanosomes, enabling animals to appear lighter or darker for camouflage, thermoregulation, and UV-protection. A complex series of hormonal and neural mechanisms regulates melanophore pigment distribution, making these cells a valuable tool to screen toxicants as a dynamic cell type that responds rapidly to the environment. We found that maltol, a naturally occurring flavor enhancer and fragrance agent, induces melanophore pigment aggregation in a dose-dependent manner in Xenopus laevis tadpoles. To determine if maltol affects camouflage adaptation, we placed tadpoles into maltol baths situated over either white or black background. Maltol induced pigment aggregation in a similar dose-dependent pattern regardless of background color. We also tested how maltol treatment compares to melatonin treatment and found that the degree of pigment aggregation induced by maltol is similar to treatment with melatonin, but the time course differs significantly. Last, maltol had no effect on mRNA expression of pro-opiomelanocortin or melanin concentrating hormone receptor in the brain, both of which regulate camouflage-related pigment aggregation. Our results suggest that maltol does not exert its effects via the camouflage adaptation mechanism nor via melatonin-based mechanisms. These results are the first to identify a specific toxicological effect of maltol exposure and rules out several mechanisms by which maltol may exert its effects on pigment aggregation.

Toxcast/Tox21 is a massive federally run research effort dedicated to better understanding the potential toxicity of thousands of compounds in a high throughput manner. Among this list of compounds is equilin, an estrogen-like compound that was flagged as a potential thyroid hormone agonist. Here we examine if equilin acts like a thyroid hormone agonist on cellular and molecular mechanisms of brain development in Xenopus laevis tadpoles. To examine the effect of equilin, tadpoles were divided into eight groups and received 4 days of exposure. The experimental groups were as follows: 1 µL, 10 µL, and 100 µL of equilin, 1 µL, 10µM, and 100 µM of 17-β estradiol as an estrogen control, 15 µg/mL thyroxine (T 4 ) as a thyroid hormone control, and a no-exposure control. After 4 days of treatment, animals were treated with CldU to label dividing cells for 2hr and then euthanized in MS-222. After fixation, body length was measured and the brains dissected out. IHC was performed on brains for CldU to label proliferating neural progenitor cells. Brains were then whole-mounted and analyzed using confocal microscopy. We found that equilin did not increase the number of dividing progenitor cells in a T 4 -like manner. Instead, equilin decreased proliferation in a dose-dependent manner, as did estradiol. The same paradigm was performed separately staining for caspase-3 and h2ax, finding that equilin increased cell death in contrast to CNTL and T 4 . In another experiment, RNA was extracted from tadpole brains in each group and qPCR was performed to assess change in expression of thyroid hormone-sensitive genes, Equilin did not affect gene expression in a thyroid hormone-like manner. Our data indicate that equilin does not act as a thyroid hormone agonist in the Xenopus laevis nervous system but instead acts similarly to estradiol. Our data strongly suggest that equilin is not a TH disruptor, contrary to the findings of the ToxCast/Tox21 dataset.

Mining, whether current or inactive, generally increases salt concentrations in catchment watersheds due to precipitation on and through exposed rock surfaces. Practices like mountaintop removal mining have exacerbated this issue, with measurements of salt concentrations in nearby catchment systems well above normal levels. Nevertheless, the impact of the ionic composition of mining effluent on aquatic animal health is not well understood. This is a particularly important issue in Appalachia because it is home to an enormous diversity of organisms, including a huge array of amphibians that live in streams that receive mining effluent from operating and abandoned mines. To investigate this issue, we examined the effects of reconstituted mining effluent on the development of wild-caught wood frog (Lithobates sylvaticus) tadpoles. We collected day-old fertilized eggs from a creek near Blacksburg, VA in early March, 2018 and raised them to hatch. Tadpoles were then assigned to either sulfate or chloride-based reconstituted mining effluent diluted to six different conductivities (100 µS/cm -2,400 µS/cm). After 7 or 14 days of treatment, tadpoles were euthanized and fixed in paraformaldehyde. We imaged the heads and bodies of tadpoles for morphometric analysis before dissecting out brains and immunostaining them for phospho-histone H3, which labels dividing progenitor cells in the brain. We found that sulfate-based reconstituted mining effluent significantly lowered progenitor cell division at 1200 µS/cm at Day 7 and at 600 µS/cm at Day 14 relative to control. Chloride-based reconstituted mining effluent was less impactful, with no significant differences observed at Day 7 and significantly lowered progenitor cell division at 2400 µS/cm at Day 14. In addition, both treatments slowed growth of some head morphological features, including head size and interocular distance. Chloride treatment slowed growth of body length at Day 14 at 600 µS/cm, whereas sulfate-based reconstituted mining effluent had no effect on body length. These data show that sulfate-based mining effluent has a substantial

Lead (Pb) poisoning during early development is associated with behavioral and cognitive deficits, but the specific mechanisms by which Pb impairs brain development are not fully understood. One potential mechanism is that Pb poisoning may impair thyroid hormone (TH)-mediated changes in brain development. To address this issue, we performed experiments to assess the effects of Pb poisoning on (TH)-dependent changes in cellular and molecular mechanisms in the developing Xenopus laevis tadpole brain. We treated stage 48 tadpoles to combinations of 1000 ppb Pb bath for seven days and added one of three different concentrations of thyroxine (T4) for the final two days of treatment. We found that lead exposure decreased body length, including in T4-treated tadpoles. We also performed immuno-staining for proliferative marker pH3 and found that Pb disrupts T4-induced increases in neuronal proliferation. Finally, we used syGlass VR data visualization software to measure volume of the forebrain, midbrain, and hindbrain in 3D and found that Pb exposure impaired T4- mediated changes in brain volume. Last, we found that Pb poisoning reduced the T4-mediated increase in proliferating cell nuclear antigen (PCNA), a TH-sensitive gene. These results illustrate that Pb poisoning impairs some TH-dependent changes in the developing brain.