U.S. Environmental Protection Agency, Mid-Continent Ecology Division, 6201 Congdon Boulevard, Duluth, Minnesota 55804
Received April 3, 2000; accepted June 13, 2000
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ABSTRACT |
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Key Words: all-trans retinoic acid (RA); hindlimb malformation; Rana clamitans; Rana pipiens; Rana septentrionalis; Rana sylvatica; Xenopus laevis.
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INTRODUCTION |
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Retinoids are metabolic derivatives of vitamin A that act as signaling molecules, and regulate many processes critical to early embryonic development including axial patterning, cellular differentiation, tissue induction, proliferation, and apoptosis (Sucov and Evans, 1995). Retinoids elicit their effects through binding to members of the steroid-thyroid nuclear receptor superfamily and regulating transcription of specific target genes. To date, two distinct families of retinoid-responsive nuclear receptor transcription factors have been characterized, the retinoic acid receptors (RARs) and retinoid x receptor (RXRs) (Sucov and Evans, 1995
). These receptors are present in all vertebrates studied, share considerable homology, and have similar spatial-temporal patterns of gene expression during development across species (Sucov and Evans, 1995
). Alterations in retinoid levels, whether conditions of excess or deficiency, result in developmental anomalies in nearly all vertebrate species studied.
The role of retinoids in vertebrate limb development has received considerable attention. When administered to regenerating amphibian limbs, retinoids induce proximo-distal and mirror-image duplications (Maden, 1983; Thoms and Stocum, 1984
, Scadding and Maden, 1986a
,b
). In the chick, local application of retinoids affects limb pattern regulation by mimicking the effects of the zone of polarizing activity, causing anterior-posterior duplication (Summerbell 1983
; Tickle et al., 1982
, 1985
). One common feature of these amphibian and chick experiments is the employment of surgical manipulation of the limb tissue. In contrast, systemic exposure of developing limbs to retinoids does not result in limb duplication. Rather, the treatment of developing limb bud stage mice (Kochhar, 1977
, 1984), Xenopus laevis (Scadding and Maden, 1986b
) and Axolotl (Scadding and Maden, 1986a
), in the absence of physical manipulation, results in concentration and developmental stage-dependent deletions and reduction of hind-limb skeletal segments.
Due to the importance of the retinoic acid signaling system in regulating embryonic development, the presence of xenobiotic chemicals in the environment capable of disrupting this system could have important implications for amphibian development and survival. One important issue when considering the developmental toxicity of retinoids is the apparent decrease in sensitivity of the vertebrate embryo with progression through development. In mice and rats, gastrulation stage exposure to retinoids results in a broad spectrum of craniofacial defects (Sulik et al., 1988). Sulik and coworkers have shown that 1.25 mg/kg RA, given to gravid females on gestation day 7 (gastrulation stages), produces micro/anophthalamia and exencephaly (Sulik et al., 1995
). These malformations are not conducive to life as a neonate, but the embryo will often develop to term. Studies conducted in rats have shown that RA, administered at 25 mg/kg as a single dose during gastrulation, results in near 100% embryo lethality and resorption by gestation day 12 (Vickers, 1985
). With progression of the mammalian embryo into organogenesis, there is a decrease in the sensitivity to retinoids. Experiments in mice have shown that RA concentration must be increased to 50 mg/kg body weight of the dam to produce limb dysmorphogenesis in gestation day 11 embryos (Kochhar et al., 1984
). At this stage of development an RA concentration of 100 mg/kg produced only 12% embryo lethality. In amphibians, there are data on the effects of retinoids on gastrulating and neurulating embryos (Dawid et al., 1993
; Creech Kraft and Juchau, 1995
; Creech Kraft et al., 1995
; Leroy and De Robertis, 1992
) and on hind-limb development and regeneration (Maden, 1983
, 1985
; Scadding and Maden, 1986). However, the experimental work has not been systematic enough to clearly assess stage-dependent sensitivity to retinoids.
Ideally, native Ranid species would be used to evaluate the potential for environmental retinoid mimics to affect embryonic and larval development. However, conditions for extended culture and breeding of Ranid species are not well defined, and the use of field-collected embryos is limited to the natural breeding season. As an alternative, X. laevis is a well-studied model of vertebrate development for which a great deal of molecular data are available. Its culture is routine, reproduction is not limited to natural breeding seasons, and conditions for toxicological testing are standardized (ASTM 1999). To date, there have been no reports regarding the comparative developmental toxicity of retinoids in X. laevis and native Ranid species.
In the experiments described herein, we compare the developmental toxicity of RA in mid-blastula and/or limb bud stage X. laevis, and 4 native Ranid species (R. clamitans (green frog, R. septentrionalis (mink frog), R. pipiens (northern leopard frog), and R. sylvatica (wood frog)). RA was used because it is a potent ligand for the retinoic acid receptors and is the standard retinoid to which nearly all others are compared. Our experiments provide information as to the feasibility and potential limitations associated with using X. laevis as a model organism to study the effects of potential retinoid exposure in wild populations of amphibians and to address the issue of stage-specific and species-specific sensitivity. Further, we provide the first report of the developmental toxicity of RA in R. clamitans, R. septentrionalis, R. pipiens, and R. sylvatica.
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MATERIALS AND METHODS |
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Embryo Exposures
Rana septentrionalis and R. clamitans were staged according to Gosner (1960) and X. laevis were staged according to Nieuwkoop and Faber (1994). Table 1 is a comparison of the two staging systems at developmental stages relevant to the experiments described below. Stage 8 R. septentrionalis, R. clamitans, and X. laevis embryos were exposed to the solvent control (0.08% ethanol), 6.25, 12.5, 25, and 50 ng RA/ml in Lake Superior water. Each treatment group consisted of two replicates of 40 randomly selected embryos each. The static exposures were conducted in 50-ml glass beakers containing 25 ml of test solution, at 22°C under conditions of low light for 24 h. At test initiation (0 h), exposure solutions were analyzed for RA concentrations by high pressure liquid chromatography (HPLC) (Table 2
). After 24 h of exposure, embryos were moved to clean water and maintained as described above.
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Tadpole Exposures
Stage 28 R. sylvatica, stage 28 R. clamitans, stages 26 and 28 R. pipiens, and stages 48 and 51 X. laevis tadpoles were exposed to solvent control (0.05% ethanol), 250, 500, 750, 1000, and 1250 ng/ml RA in Lake Superior water, under conditions of low light. The R. pipiens, R. sylvatica, and R. clamitans treatment groups consisted of 2 replicates of 10 randomly selected organisms per test chamber. The X. laevis treatment groups consisted of 2 replicates of 13 organisms per test chamber. In these experiments, the volume of test solution was varied to maintain a consistent mass to volume ratio across all species. Aliquots of the exposure solution were collected at 0 h and analyzed by HPLC to determine initial RA concentrations. The data are presented as nominal concentrations. Additional water samples were collected at 4, 12, and 24 h in the R. clamitans and stage 26 R. pipiens experiments to determine the rate of loss of RA from exposure solutions. After the 24-h exposures, tadpoles were moved to clean water and maintained until forelimb emergence, as described above. At stages 45 (Ranid) and 63 (X. laevis), tadpoles were anesthetized with tricaine methanesulfonate, evaluated for external malformations, and fixed in 95% ethanol. Tadpoles were stained with alcian blue and alizarin red, cleared, and examined for gross abnormalities of bone and cartilage (method described below).
Bone and Cartilage Staining Procedure
Tadpoles were fixed in 95% ethanol and stored in this solution. They were then transferred to distilled water to soften the skin prior to removal. Skinned tadpoles were washed in 70% ethanol for at least 1 h and then stained in alcian blue staining solution (per liter: 90 mg alcian blue from Sigma, 600 ml absolute ethanol, and 400 ml glacial acetic acid) for 48 h. Tadpoles were washed x3 in distilled water and digested in 3% potassium hydroxide for 26 h, depending on the size of the organisms. Bone was stained by placing tadpoles in alizarin red (per liter: 35 mg potassium hydroxide, 200 ml glycerin, 75 mg alizarin red (Sigma), and 800 ml H2O ) for 24 h. Tadpoles were cleared through graded glycerin solutions and stored in 100% glycerin until hind limbs were evaluated.
Water Analysis
Water samples (0.5 ml) collected from embryo and tadpole exposures were placed into amber vials containing methanol (0.5 ml) and immediately analyzed for RA, using a Hewlett-Packard 1050 HPLC (Avondale, PA) equipped with a diode-array detector (wave length 354 nm) and a temperature-controlled (7°C) autosampler. An aliquot of the sample (300 µl) was injected directly onto a Nucleosil C18 AB column (Alltech, Deerfield, IL), and the column eluted with a 40 mM ammonium acetate buffer (pH 7.3) and methanol gradient program at a flow rate of 1 ml/min. Data were collected with Hewlett-Packard ChemStation software (version A.05.01), and RA concentrations determined using the external standard method of quantification with a 7-point linear calibration curve. Routine (i.e., 10%) quality assurance analyses (matrix blanks and spikes) were conducted with each sample set. No RA was detected in the blanks (n = 10) and a mean (± SD, n = 10) recovery of RA in the spiked water samples was 93 ± 5%. The analytical detection limit was 9 ng/ml.
Data Analysis
Mortality and dysmorphogenesis data were assessed using one-way ANOVA, followed by Dunnet's post hoc test. Percentage data were arcsine-square root transformed before analysis. The results were considered significant at p 0.05. Statistical analyses were performed using SYSTAT 7.0 Windows (SPSS, Chicago, IL). Data are generally discussed as a function of nominal test concentrations. However, EC50 values for dysmorphogenesis and LC50 values for the blastula stage X. laevis, R. clamitans, and R. septentrionalis exposures were calculated using measured RA concentrations at test initiation (Table 2
). Probit analyses were performed using SAS (The SAS System for Windows, Release 6.12, SAS Institute Inc., Cary, NC). Cumulative probability of survival was determined as described by Kaplan (1958).
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RESULTS |
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Rana pipiens.
Rana pipiens tadpoles were exposed at either stage 26 or stage 28 to a range of RA concentrations. RA was acutely toxic to stage 26 tadpoles, with the highest concentration (1250 ng/ml) causing 100% mortality within the first 24 h. In contrast, exposure at stage 28 did not result in increased mortality (data not shown), thus indicating a decrease in sensitivity of the tadpoles to RA with increasing age. Treatment of R. pipiens with RA did not result in hind-limb malformations in tadpoles exposed at either stage 26 or 28 (Table 4).
Xenopus laevis.
To determine the effects of RA on hind-limb development, tadpoles were exposed to RA at stage 48 or stage 51. Treatment did not result in hind-limb malformation in tadpoles exposed at stage 48 (Table 4). In contrast, RA exposure resulted in a concentration-dependent increase in frequency and severity of hind-limb malformations in tadpoles exposed at stage 51 (Fig. 5
). When examined grossly, limbs from exposed organisms appeared to have either shortened or missing skeletal elements. Examination of cleared limbs revealed severe bending at the diaphysis of the bone such that the opposite heads of the bone fused to formed a triangular structure with the diaphysis of the fused segments forming the apex of the triangle (Fig. 6b
). At 250 and 500 ng RA/ml, typically only one of the hind-limb segments was affected (Fig. 6b
). Conversely, at 750, 1000, and 1250 ng RA/ml, 2 to 4 hind-limb segments were affected (Fig. 6c
). In addition to the triangular malformations, RA treatment resulted in total loss of limb segments at 1250 ng RA/ml (data not shown).
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DISCUSSION |
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Duplication of posterior structures, including the pelvis and hind limb, have been reported in mice following retinoid exposure at the blastula stage of development (Rutledge et al., 1994). To determine if this type of effect would occur in amphibians, embryos were exposed to a pulse of RA at mid-blastula stages, maintained in clean water, and evaluated for hind-limb malformations when the forelimbs emerged. In the 3 species tested, RA exposure at sub-lethal concentrations did not result in abnormalities or duplication of posterior structures, including the hind limbs. The apparent difference in the way mammalian versus amphibian embryos respond to RA is not surprising given inherent differences in reproduction and development in these 2 classes. In mammals, exposure of the early embryos results in craniofacial malformations, which are not conducive to life as a neonate; however, affected embryos will develop to term (Rutledge et al., 1994
; Sulik et al., 1988
, 1995
). This is possible because the embryo receives the necessary nutrition to develop through organogenesis from the maternal blood flow and, as a consequence, effects on posterior structures are manifested and readily observable. However, in amphibians, development through metamorphosis and, ultimately, manifestation of posterior malformations are dependent on the early tadpole feeding, normally. In our experiments, there was a good correlation between the percentage of embryos exhibiting dysmorphogenesis just after hatching and the percent dead by the earliest limb bud stage (Figs. 24
). This suggests embryos that suffered dysmorphogenesis ultimately died once the yolk was completely adsorbed. This was likely due to the need for tadpoles to acquire their own nutrition through feeding, which is greatly impaired by severe anterior dysmorphogenesis. Our data suggest that anterior structures are more sensitive to RA exposure than posterior structures and that concentrations high enough to produce posterior effects are not conducive to life as a tadpole. Consequently, posterior malformations are never manifested following exposure at the mid-blastula stage.
Retinoids have been shown to have the ability to mimic the effects of the zone of polarizing activity in developing limbs, causing anterior-posterior duplication (Summerbell, 1983; Tickle et al., 1982
, 1985
). When administered to regenerating amphibian limbs, retinoids induce proximo-distal and mirror-image duplications (Maden, 1983
; Scadding and Maden, 1986a
,b
; Thoms and Stocum, 1984
). Given the ability of retinoids to alter normal limb patterning, it is not surprising that systemic exposure of developing limb-bud stage mice (Kochhar, 1977
, 1984), X. laevis (Scadding and Maden, 1986b
), and axolotl (Scadding and Maden, 1986a
) results in concentration- and developmental stage-dependent deletions and reductions of hind-limb skeletal segments. In our experiments, RA caused hind-limb malformations in stage 51, but not in stage 48 in X. laevis. It is reasonable to suggest that apparent differences in sensitivity between stage 48 and stage 51 X. laevis hind limbs are a consequence of the early limb-bud tissue being in a less active developmental state. It is possible that if a longer duration and/or multiple pulse exposure was used, hind limbs would have been affected in stage 48 tadpoles. Similar to stage 51 X. laevis, stage 28 R. sylvatica tadpoles were also sensitive to RA-induced hind-limb malformation. These malformations consisted mainly of reductions in one or more of the limb segments. Examination of cleared limbs revealed 2 types of malformations to the limb segments. In the most severely affected organisms, entire limb segments failed to develop. In less severely affected organisms, a second type of malformation was observed involving bending of the bone at the diaphysis and fusion of the bone to form a triangular structure. These malformations are similar to those reported previously for stage 51 X. laevis following retinoid exposure (Scadding and Maden, 1986a
).
As with stage 48 X. laevis, RA was ineffective in inducing hindlimb abnormalities in stage 26 and 28 R. pipiens tadpoles, and stage 28 R. clamitans. The highest concentration of RA tested in these experiments produced 100% mortality in stage 26 R. pipiens tadpoles. This indicates that a pulse exposure of RA at this stage is developmentally toxic to R. pipiens tadpoles, but not teratogenic. Similarly, in the chick, systemic exposure to retinoic acid had no effect on the limb at concentrations that were not embryo lethal (Summerbell and Harvey, 1983). The apparent disparity in responses among stage 28 R. clamitans, R. pipiens, and R. sylvatica may be explained by the differences in the rate at which the 3 species develop. In the time frame of these experiments, R. sylvatica developed nearly twice as fast as R. pipiens and R. clamitans. Thus, given the fact that RA rapidly degraded in aqueous solution, R. sylvatica tadpoles were exposed over a longer developmental period. In support of this hypothesis, X. laevis and R. sylvatica, which have similar developmental rates, responded similarly to RA exposure. This highlights the limitations associated with using static exposure systems for testing compounds that degrade rapidly in aqueous solution. To more accurately assess highly labile compounds in species with such different developmental rates, a continuous exposure system is necessary.
In amphibians, there are data on the effects of retinoids on the early embryo (Dawid et al., 1993; Creech Kraft and Juchau, 1995
; Creech Kraft et al., 1995
; Leroy and De Robertis, 1992
) and during hind-limb development (Maden, 1983
; Maden, 1985
; Scadding and Maden, 1986a
,b
). However, previous experiments have not systematically assessed stage-dependent sensitivity to retinoids. We have conducted mid-blastula and limb bud stage exposures in R. clamitans and X. laevis to generate the data necessary to evaluate the stage-specific sensitivity of RA in these two species. Exposure to RA at early limb-bud stages had little effect on limb development when tested at 50 to 100 times the concentrations necessary to produce dysmorphogenesis and increased mortality, respectively, at earlier life-stages. The wealth of information regarding the stage sensitivity of RA in rodents indicates a similar decrease in sensitivity to RA-induced teratogenesis and lethality, with progression of the mammalian embryo through development.
It has been proposed that hind-limb malformation observed in amphibians across North American may be attributed to xenobiotic disruption of retinoid signaling pathways (Gardiner and Hoppe, 1999). This proposal is based on the phenotypes observed in R. septentrionalis specimens collected from an unnamed lake in central Minnesota. Malformations included reductions, deletions, and supernumerary structures. Retinoids have been shown to induce limb duplications in avian and amphibian species (Maden, 1983
; Scadding and Maden, 1986a
,b
; Summerbell 1983
; Thoms and Stocum, 1984
; Tickle et al., 1982
, 1985
). However, this is only after employment of surgical manipulation of the limb tissue. The only report of systemic retinoid exposure causing limb duplication was following a blastula-stage exposure in mice (Rutledge et al., 1994
). We have presented data that indicate anterior structures are more sensitive to RA exposure than posterior structures. Concentrations of RA high enough to produce posterior effects are lethal to tadpoles, and consequently, limb duplications are not observed. Thus, available laboratory data indicate it is unlikely that limb duplication observed in field specimens are a consequence of disruption of retinoid pathways.
This leaves the possibility that xenobiotic disruption of retinoid signaling pathways may be the cause of deletions and reduction in hind limbs of amphibians from the field. The phenotypes described for X. laevis by Scadding and Maden (1986b) and in this report are very similar to those described for native anurans (Gardiner and Hoppe, 1999). Further, we have shown that laboratory RA exposure induces a similar phenotype in R. sylvatica. However, it is premature to speculate that malformations observed in the field are a consequence of disruption of retinoid pathways based on phenotypes alone. Additionally, there is an important observation highlighted by our studies, which contradicts the notion that retinoid mimics in the environment are responsible for hind-limb malformations. Our data and several mammalian studies indicate that the early vertebrate embryo is considerably more sensitive to retinoid excesses than limb-stage organisms. Furthermore, our study demonstrates that RA causes embryo lethality at much lower concentrations than those necessary to cause reductions and deletions of the hind limb. Under continuous exposure scenarios, embryos would not survive to develop to stages sensitive to chemicals that elicit limb malformation through the retinoid signaling pathways. This suggests that if retinoid mimics in the environment are causing hind-limb malformations, this would only occur under scenarios of pulsed-chemical exposures. The pulse exposure scenario is possible, but observations are often reported for multiple anuran species, which have different breeding periods and developmental rates, from the same affected sites. It is difficult to put forth a pulse exposure scenario that would expose one species at the correct developmental stage to induce limb malformations without exposing other species at embryonic stages. For example, R. pipiens are late-spring spawners, while R. clamitans and R. septentrionalis are midsummer spawners. Due to this natural spawning behavior, R. clamitans and R. septentrionalis are at embryonic stages when R. pipiens reach the sensitive stages for induction of hind-limb malformations. Consequently, if retinoid mimics were present at concentrations high enough to induce limb malformation in R. pipiens, they would have dramatic effects on the survival of R. clamitans and R. septentrionalis embryos. To more clearly eliminate the possibility that disruption of retinoid signaling can produce the spectrum of hind-limb malformation observed in the field, a chronic, continuous exposure should be conducted to demonstrate the stage-specific sensitivity using an exposure which would more closely model an environmental exposure.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (218) 529-5003. E-mail: degitz.sigmund{at}epamail.epa.gov.
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