Dietary antioxidants enhance immunocompetence in larval amphibians.

Dietary antioxidants have been shown to confer a variety of benefits through their ability to counter oxidative stress, including increased immunocompetence and reduced susceptibility to both infectious and non-infectious diseases. However, little is known about the effects of dietary antioxidants on immune function in larval amphibians, a group experiencing worldwide declines driven by factors that likely involve altered immunocompetence. We investigated the effects of dietary antioxidants (quercetin, vitamin E, and ß-carotene) on two components of the immune system, as well as development and growth. Lithobates pipiens tadpoles fed diets with supplemental ß-carotene or vitamin E exhibited an enhanced swelling response as measured with a phytohemagglutinin assay (PHA), but there was no induced antibody response. Effects were often dose-dependent, with higher antioxidant levels generally conferring stronger swelling that possibly corresponds to the innate immune response. Our results indicate that the antioxidant content of the larval amphibian diets not only had a detectable effect on their immune response capability, but also promoted tadpole growth (mass gain), although developmental stage was not affected. Given that many environmental perturbations may cause oxidative stress or reduce immunocompetence, it is critical to understand how nutrition may counter these effects.

Copper and nickel effects on survival and growth of northern leopard frog (Lithobates pipiens) tadpoles in field-collected smelting effluent water.

Trace metals can have subtle yet chronic impacts on organisms by inducing physiological stress that reduces their survival or impedes their ability to tolerate additional environmental stressors. The toxicity literature indicates, however, that aquatic organisms react differently to trace metals depending on the environments in which they reside. The objective of the present study was to understand the response of northern leopard frog (Lithobates pipiens) larvae to ionic copper (Cu), nickel (Ni), and their combination within an effluent water collected downstream of a tailings wetland area. Tadpoles were assigned randomly to 1 of 8 Cu concentrations (8-200 µg/L), 7 Ni concentrations (160-1200 µg/L), or 8 Cu and Ni combined concentrations (8:160-200:1200 µg/L) and showed significant differences in survival and life history traits among treatments. In the Cu and Cu and Ni combined treatments, tadpole survival decreased with increased Cu exposure starting at Cu = 160 µg/L and in the Ni treatment, tadpole survival decreased with increased Ni exposure starting at Ni = 650 µg/L. All Cu-exposed treatments induced a growth increase as the concentration increased, whereas the tadpoles showed a significant decrease in growth rate in Ni treatments. These contrasting outcomes suggest a plastic response to trace metals whereby tadpoles allocate energy reserves toward either escaping or coping with stress. Finally, the authors' argue that future studies will benefit from examining the impacts of multiple stressors in aquatic ecosystems to provide better environmental mitigation.

Immunomodulation in post-metamorphic northern leopard frogs, Lithobates pipiens, following larval exposure to polybrominated diphenyl ether.

Pollutants and disease are factors implicated in amphibian population declines, and it is hypothesized that these factors exert a synergistic adverse effect, which is mediated by pollutant-induced immunosuppression. Polybrominated diphenyl ethers (PBDEs) are ubiquitous pollutants that can exert immunotoxicity, making them of interest to test effects on amphibian immune function. We orally exposed Lithobates (Rana) pipiens tadpoles to environmentally realistic levels (0-634 ng/g wet diet) of a pentabromodiphenyl ether mixture (DE-71) from as soon as they became free-swimming through metamorphic climax. To assess adaptive immune response in juvenile frogs, we used an enzyme-linked immunosorbent assay to measure specific IgY production following immunization with keyhole limpet hemocyanin (KLH). Specific KLH antibody response was significantly decreased in juvenile frogs that had been exposed to PBDEs as tadpoles. When assessing innate immune responses, we found significantly different neutrophil counts among treatments; however, phagocytic activity of neutrophils was not significantly different. Secretion of antimicrobial skin peptides (AMPs) nonsignificantly decreased with increasing PBDE concentrations, and no significant effect of PBDE treatment was observed on efficacy of AMPs to inhibit chytrid fungus (Batrachochytrium dendrobatidis) growth. Our findings demonstrate that environmentally realistic concentrations of PBDEs are able to alter immune function in frogs; however, further research is needed to determine how these alterations impact disease susceptibility in L. pipiens.

Effects of chronic polybrominated diphenyl ether exposure on gonadal development in the northern leopard frog, Rana pipiens.

Polybrominated diphenyl ethers (PBDEs) are bioaccumulative, persistent organic pollutants used as flame retardants in consumer goods. Concentrations of PBDEs in North American wildlife have been increasing for decades and been shown to have estrogenic effects on sexual development. No studies, however, have examined the effects of PBDEs on the sexual development of North American frogs at ecologically relevant concentrations. This study examined the effects of five dietary concentrations of DE-71 (0, 1.1, 6.1, 71.4, and 634 ng ΣPBDEs/g diet), a technical PBDE mixture, on the gonadal development of the northern leopard frog, Rana pipiens. Tadpoles were exposed chronically from the time they became free-swimming until metamorphosis. Frogs were killed either at metamorphic climax or 10 weeks after completing metamorphosis, processed for histology, and examined for alterations in sexual development. The experimental group exposed to PBDEs at 1.1 ng/g had a significantly larger proportion of females compared with the expected 50:50 sex ratio. At 10 weeks post-metamorphosis, male frogs exposed to 6.1 and 71.4 ng/g had significantly smaller testes, but all other measure of gonadal development tested showed no effects. No intersex or increased incidence of gonadal abnormality were detected. These findings indicate that PBDEs may disrupt sexual differentiation in frogs at low, environmentally relevant concentrations.

Developmental profiles and thyroid hormone regulation of brain transcripts in frogs: a species comparison with emphasis on Physalaemus pustulosus.

In amphibians, thyroid hormones (THs) are considered key regulators of brain remodeling during metamorphosis, while sex steroids (estrogens and androgens) control sexual differentiation and gonadal development. However, these two endocrine axes can interact during tadpole brain development. Previously, we demonstrated that THs affect sex steroid-related gene expression in the developing brain of Silurana tropicalis and Rana pipiens; however, the gene expression changes differed between species. We chose to study a third anuran species, Physalaemus pustulosus, to test new hypotheses about the role of THs in the regulation of brain gene expression. We first established developmental transcript profiles of TH- and sex steroid-related genes in the brain of P. pustulosus. Then, following the same protocols as in our previous studies, we investigated triiodothyronine (T3) regulation of brain transcripts in premetamorphic P. pustulosus and then compared the results with our previous two studies. In the case of TH-related genes, TH receptor beta (trbeta) and deiodinase type 3 (dio3), mRNA developmental profiles were similar in the three species and with respect to other species in the published literature. However, the profiles of TH receptor alpha (tralpha) and deiodinase type 2 (dio2) mRNA revealed differences between anuran species. Among the three anurans we have studied, the direction of the T3 regulation of TH-related genes was overall similar, but the magnitude of gene expression change differed depending on the rate of metamorphosis in a given species. For the sex steroid-related genes, each species exhibited similar developmental profiles but differed in their response to T3. In P. pustulosus, T3 reduced the expression of aromatase (cyp19) while increasing mRNA levels of androgen and estrogen receptors. These results are similar to previous research in R. pipiens but differ from data for S. tropicalis, for which we found an increase in androgen synthesis enzymes but no effect on cyp19. Together, we propose that T3 has the potential to induce the brain androgen system in anurans. This could be achieved by increasing androgen synthesis enzymes (S. tropicalis) or by decreasing estrogen synthesis (due to a decrease in cyp19 in P. pustulosus and R. pipiens). In conclusion, we demonstrated that mechanisms of hormone interactions differ between anuran species, but in all cases T3 appears to affect the balance of sex steroids in the brain, stimulating the androgen system. We have shown that the regulation of sex steroid-related genes by T3 is more similar among closely related species than species with similar reproductive and developmental characteristics.

Apoptosis in the digestive tract of herbivorous Rana pipiens larvae and carnivorous Ceratophrys ornata larvae: an immunohistochemical study.

The lifespan of herbivorous Rana pipiens larvae is ∼3 months, while that of carnivorous Ceratophrys ornata larvae is only about 2 weeks. During metamorphic climax, the larval gut shortens dramatically, especially in R. pipiens, and its luminal epithelium is replaced by adult-type epithelium. To determine when programmed cell death occurs during the metamorphic restructuring of the gut, we prepared cross-sections of the stomach, small intestine, and large intestine from representative larval stages and from juvenile frogs of both species. The sections were incubated with monoclonal antibody against active caspase-3, one of the key enzymes in the apoptotic cascade. We observed apoptosis in some luminal epithelial cells in each of the three regions of the larval gastrointestinal tract of both species. However, apoptotic cells appeared earlier in larval stages of R. pipiens than C. ornata and few were seen in juvenile frogs of either species. The results demonstrate the occurrence of apoptosis in the metamorphic remodeling of the gut of both R. pipiens larvae and C. ornata larvae.

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.

The developmental effects of a municipal wastewater effluent on the northern leopard frog, Rana pipiens.

Wastewater effluents are complex mixtures containing a variety of anthropogenic compounds, many of which are known endocrine disruptors. In order to characterize the developmental and behavioral effects of such a complex mixture, northern leopard frogs, Rana pipiens, were exposed to a range of concentrations (0%, 10%, 50%, 100%) of municipal wastewater effluent from the egg stage through metamorphosis. The estrogenic activity of the effluent was quantified by the calculation of an estradiol (E2) equivalency (EEQ) factor and was determined to be equivalent to 1.724+/-2.103ng/L E2. Individuals from the 50% and 100% wastewater treatments took significantly longer to reach metamorphosis than individuals in the 0% and 10% treatments. An increased incidence of male testicular oocytes was observed in the 50% and 100% treatments when compared to the control treatment. Morphological changes in the thyroid glands of 100% wastewater-treated individuals were also noted. No effects of wastewater exposure on growth, sex ratio, swim speed, startle response, or female gonadal development were observed. These results suggest that municipal wastewater effluent can alter the timing of the metamorphic process and impact male sexual development in R. pipiens.

Chronic exposure of Rana pipiens tadpoles to UVB radiation and the estrogenic chemical 4-tert-octylphenol.

While adverse effects of exposure to ultraviolet B radiation (UVBR) and environmental pollutants on amphibians have been documented, few studies examined the effects of interactions between environmental stressors on amphibian health. Here the impacts of chronic exposure to UVBR and the estrogenic chemical 4-tert-octylphenol (OP), alone and in combination, on the survival, development, growth, and metamorphosis of Rana pipiens (northern leopard frog) tadpoles are evaluated. Tadpoles were exposed to environmentally relevant levels of OP (0.01 or 10 nM), with and without exposure to UVBR (approximately 0.22 W/m2). After 8 mo of exposure, animals were transferred to a clean-water grow-out system for 3 additional mo. There was no effect of treatments on the weights of animals at wk 42 or on the age, snout-vent length (SVL), and weights of tadpoles at forelimb emergence (FLE). However, tadpoles exposed to UVBR had markedly delayed development and significantly fewer reached metamorphosis compared to control animals. Although exposure to UVBR and OP together produced the highest incidence of mortality and developmental anomalies, UVBR alone was sufficient to produce significant mortality of tadpoles, and exposure to UVBR or OP alone induced a significant increase in malformations and abnormalities. The malformations observed consisted mainly of spinal curvature. Our results show that chronic exposure to environmental levels of UVBR or OP alone exerts lethal and/or sublethal effects on R. pipiens tadpoles.

Effect of mono-ortho and di-ortho substituted polychlorinated biphenyl (PCB) congeners on leopard frog survival and sexual development.

We tested the effect of mono-ortho and di-ortho PCB congeners on northern leopard frog (Rana pipiens) hatching success, survival and sexual development. Embryos and tadpoles were exposed to two levels (0.5 and 50 microg/l) of two PCBs. PCBs 101 and 70 were selected because they were present in amphibians collected in the Fox River-Green Bay ecosystem and they have the theoretical structural requirements to be able to bind to the estrogen receptor and mediate estrogenic responses. The exposure of leopard frog embryos and tadpoles to PCB 70 and 101 did not significantly affect hatchability, survival, deformities or growth. There were significant departures from the expected 50:50 sex ratio in tadpoles/froglets exposed to PCB 101 and PCB 70. In all the cases of significant departure, the bias was towards higher number of females. Decrease in the proportion of male gonads and increase in the proportion of intersex gonads were observed with increasing PCB tissue concentrations. The effects of PCB congeners on sexual differentiation occur at concentrations higher than observed in frogs in the Fox River/Green Bay ecosystem.

Lethal and sublethal effects of chronic cadmium exposure on northern leopard frog (Rana pipiens) tadpoles.

Data on the chronic effects of cadmium on amphibians are lacking in spite of widespread anthropogenic contamination of terrestrial and aquatic systems. We exposed embryos and tadpoles of northern leopard frogs (Rana pipiens) to Cd (control, 0.25, 5.0, and 20.0 microg/L as CdCl2, nominal concentrations) in a static renewal system from embryonic stages to complete tail resorption. Survival of embryos (Gosner stage [GS] 17-25) was greater than 97% in all treatments. Tadpole survival was negatively correlated with dose and was significantly reduced in the 5.0 and 20.0 microg/L treatments compared with controls. Tadpole survival was greater than 80% through GS 42, forelimb emergence, for all other treatments. Tadpoles exposed to 0.25 and 5.0 microg/L exhibited increased growth by week 11; tadpoles exposed to 5.0 microg/L were significantly younger at forelimb emergence. Whole-tadpole body burdens of Cd were positively correlated with increasing Cd treatments. Cadmium was shown to alter growth and development in a native amphibian species at ecologically relevant concentrations. The existing chronic water quality criterion for Cd appears to be protective of amphibians. However, additional studies with other chemicals are needed to further explore the potential for adverse effects of contaminants on the complex life cycle of amphibians.

Hormone cross-regulation in the tadpole brain: developmental expression profiles and effect of T3 exposure on thyroid hormone- and estrogen-responsive genes in Rana pipiens.

During metamorphosis, the tadpole neuroendocrine brain is a major target for the organisational effects of hormones acting via both endocrine feedback mechanisms and local hormone production. While the receptor-mediated actions of thyroid hormones in brain development have been well described, there is evidence that thyroid hormones could also be an important modulator of estrogen action during metamorphosis. To better understand hormone action and potential cross-regulation between thyroid hormone and estrogen, we examined changes in thyroid hormone receptors (TRalpha and TRbeta) and the estrogen receptor (ERalpha) in the brain of Rana pipiens throughout metamorphosis and in response to 48 h waterborne triiodothyronine (T3) exposure (0.5, 5 and 50 nM). We also measured mRNA levels of iodothyronine deiodinase (D2 and D3) and aromatase, key enzymes responsible for local synthesis and availability of thyroid hormones and estrogen, respectively. A real-time PCR strategy targeting these genes was developed using either a fluorescent dual-labelled probe- or SYBR Green I-based method. TRbeta mRNA levels were increased during development and in response to T3 exposure. Deiodinase (D2 and D3) enzymes were differentially regulated during development, but mRNA levels of both were increased with 50 nM T3 exposure. ERalpha and aromatase mRNA levels significantly increased at metamorphic climax, but whereas estrogen receptor alpha mRNA levels were increased by 50 nM T3, aromatase mRNA levels were decreased. These results (1) demonstrate that the developing amphibian brain is an important site for stage-specific thyroid hormone regulation of nuclear receptors and hormone synthesis enzymes and (2) provide the basis for further studies exploring the physiological and functional significance of the cross-regulation between thyroid status and estrogen-sensitive genes in the brain during amphibian metamorphosis.

Exposures to estradiol, ethinylestradiol and octylphenol affect survival and growth of Rana pipiens and Rana sylvatica tadpoles.

Endocrine disrupting compounds (EDCs) are often detected in the aquatic environment and can negatively affect the health of wildlife populations. However, little is known about the sensitivity of native amphibians to EDCs. Wood frogs (Rana sylvatica) and Northern leopard frogs (Rana pipiens) were exposed to three estrogenic EDCs: estradiol (E2), ethinylestradiol (EE2), and 4-tert-octylphenol (OP). In addition, R. pipiens were exposed during two developmental stages (Gosner stages 26 and 36) to examine life-stage differences in sensitivity. Tadpoles were exposed for 2 wk to 8 nominal concentrations (0.25 microM-10 microM) of each compound. Individual mortality was recorded during the exposure period, while body weight was measured at the end of 2 wk. LC50 values were calculated, and differences in body weight between vehicle control and exposed groups were assessed. Rank order toxicity of the compounds for both R. pipiens stages and both species was OP > EE2 > E2. Gosner stage 26 tadpoles were more sensitive (LC50: E2 [5.57 microM], EE2 [3.01 microM], OP [1.36 microM]) to all three compounds when compared to stage 36 tadpoles (LC50: E2 [>10 microM], EE2 [4.17 microM], OP [2.80 microM]). Interspecies comparisons revealed R. sylvatica tadpoles (LC50: E2 [2.50 microM], EE2 [1.89 microM], OP [0.74 microM]) as being more sensitive to the three compounds than R. pipiens (LC50: E2 [4.56 microM], EE2 [2.75 microM], OP [1.42 microM]). Xenoestrogen exposure also affected tadpole body weight which may have long-term adverse effects on the rate of metamorphosis. These results provide toxicological data needed for assessing sublethal effects of estrogenic compounds on amphibian development and suggest that environmental levels of OP may pose a serious risk to the health of amphibian populations.

Gonadal differentiation in frogs exposed to estrogenic and antiestrogenic compounds.

Exposure of amphibians to endocrine disrupting compounds (EDCs) may alter differentiation of gonads, especially when exposures begin during early life stages. Gonadal differentiation was observed in leopard frogs (Rana pipiens) and wood frogs (Rana sylvatica) exposed as tadpoles to estrogenic (estradiol, ethinylestradiol, nonylphenol) and antiestrogenic compounds (an aromatase inhibitor, flavone, and an antiestrogen, ICI 182780). Exposure to all compounds at micrograms/L concentrations altered gonadal differentiation in some animals by inducing either complete feminization or an intersex condition, and altered testicular tubule morphology, increased germ cell maturation (vitellogenesis), and oocyte atresia. Comparisons between the two species indicate that R. pipiens are more susceptible to sex reversal and development of intersex gonads. However, R. sylvatica also showed alterations to testicular morphology, germ cell maturation, and ooctye atresia. These laboratory results indicate that amphibians could be susceptible to altered gonadal differentiation and development when exposed to estrogenic and antiestrogenic compounds in aquatic environments, such as those impacted by agricultural, industrial, and municipal runoff.

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.

Octylphenol and UV-B radiation alter larval development and hypothalamic gene expression in the leopard frog (Rana pipiens).

We assessed octylphenol (OP), an estrogenic endocrine-disrupting chemical, and UV-B radiation, a known stressor in amphibian development, for their effects on hypothalamic gene expression and premetamorphic development in the leopard frog Rana pipiens. Newly hatched tadpoles were exposed for 10 days to OP alone at two different dose levels; to subambient UV-B radiation alone; and to two combinations of OP and UV-B. Control animals were exposed to ethanol vehicle (0.01%) exposure, a subset of tadpoles from each treatment group was raised to metamorphosis to assess differences in body weight and time required for hindlimb emergence. Tadpoles from one of the OP/UV-B combination groups had greater body weight and earlier hindlimb emergence (p < 0.05), but neither OP nor UV-B alone produced significant changes in body weight or hindlimb emergence, indicating a potential mechanism of interaction between OP and UV-B. We hypothesized that the developing hypothalamus might be a potential environmental sensor for neurotoxicologic studies because of its role in the endocrine control of metamorphosis. We used a differential display strategy to identify candidate genes differentially expressed in the hypothalamic region of the exposed tadpoles. Homology cloning was performed to obtain R. pipiens glutamate decarboxylases--GAD65 and GAD67, enzymes involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). cDNA expression profiles revealed that OP and UV-B affected the levels of several candidate transcripts in tadpole (i.e., Nck, Ash, and phospholipase C gamma-binding protein 4 and brain angiogenesis inhibitor-3) and metamorph (i.e., GAD67, cytochrome C oxidase, and brain angiogenesis inhibitor-2 and -3) brains. This study represents a novel approach in toxicology that combines physiologic and molecular end points and indicates that levels of OP commonly found in the environment and subambient levels of UV-B alter the expression of important hypothalamic genes and disrupt tadpole growth patterns.