Uptake and Metabolism of All-trans Retinoic Acid by Three Native North American Ranids

Patricia A. Kosian1, Elizabeth A. Makynen, Gerald T. Ankley and Sigmund J. Degitz

U. S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Boulevard, Duluth, Minnesota 55804

Received January 11, 2003; accepted March 25, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoids, which are Vitamin A derivatives, are important signaling molecules that regulate processes critical for development in all vertebrates. The objective of our study was to examine uptake and metabolism of the model retinoid, all-trans retinoic acid (all-trans RA), by three native North American anurans, Rana sylvatica, R. pipiens, and R. clamitans. Limb-bud stage tadpoles (stages 26–28) were exposed to all-trans RA concentrations of 0, 250, 500, 750, 1000, and 1250 ng/ml for 24 h. Water and tissue samples, collected at 0, 4, 12, and 24 h, were analyzed by high pressure liquid chromatography with diode-array detection (HPLC-DAD) to characterize aqueous exposure and all-trans RA uptake and metabolism. All-trans RA degraded rapidly in exposure water (i.e., with organisms), with over 70% of the parent compound gone in 4 h and none detected by 24 h. Consistent with this result, tadpoles from the three species showed the greatest accumulation of all-trans RA at 4 h followed by decreasing tissue concentrations at 12 and 24 h. In addition to all-trans RA, several other chromatographic peaks were observed in the tissue extracts indicating metabolism of the retinoid by the tadpoles. Identification of potential metabolites of all-trans RA and endogenous retinoids was conducted by comparing retention times and absorption spectra of available standards (i.e., 4-oxo-all-trans RA, 4-oxo-13-cis RA, 13-cis RA, 9-cis RA, all-trans retinol, all-trans retinal) to those in the tissue extracts. In all three species, all-trans RA was metabolized to 4-oxo-all trans RA and 13-cis RA. The RA isomer, 9-cis RA, was detected in two species,R. sylvatica and R. pipiens. All three species also had measurable levels of vitamin A (all-trans retinol), while the aldehyde form (all-trans retinal) was detected only in R. clamitans. Our results indicate that all-trans RA is rapidly metabolized by these Ranid species to a variety of retinoid derivatives, several of which are known ligands for RA and retinoid receptors, and are capable of activating this signaling transduction pathway.

Key Words: all-trans retinoic acid; metabolism; Rana sylvatica; Rana pipiens; Rana clamitans.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoids, which include Vitamin A and its derivatives, are low molecular weight, lipophilic molecules that are essential for a number of diverse physiological processes such as vision, reproduction, metabolism, growth and differentiation, bone development, pattern formation during embryogenesis, and overall maintenance of organism health (Mangelsdorf et al., 1994Go). Retinoic acid (RA) and possibly some of its metabolites are the active retinoid forms responsible for mediating most of the nonvisual functions of this class of compounds (Kurlandsky et al., 1995Go). The mechanism by which retinoids elicit their effects is ultimately through their ability to bind to members of the steroid-thyroid nuclear receptor superfamily and regulate gene transcription at specific target sites within the organism (Mangelsdorf et al., 1994Go; Sucov and Evans, 1995Go). Currently, two distinct families of retinoid responsive nuclear receptors have been identified and characterized: retinoic acid receptors (RARs) and retinoid receptors (RXRs), each of which include three isoforms, {alpha}, ß, {gamma} (Sucov and Evans, 1995Go). These receptors have been observed in all vertebrate classes studied (i.e., mammals, birds, and amphibians) and appear to share considerable across-species homology (Mangelsdorf et al., 1994Go; Sucov and Evans, 1995Go).

Alterations in endogenous retinoid levels, however, can result in developmental anomalies in vertebrates (Sucov and Evans, 1995Go). All-trans RA, a bioactive metabolite of vitamin A, is a particularly potent dysmorphogen with exposure causing severe malformations in developing mammals (Nau, 1993Go), as well as nonmammalian embryos including amphibians (Creech Kraft et al., 1994Go; Kratke et al., 2000Go; Pijnappel et al., 1993Go) and fish (Herrmann, 1995Go). In Xenopus laevis (often used as a representative test species for other amphibians), exposure to all-trans RA during embryogenesis produced severe multiple malformations including underdevelopment of the brain, anterior truncation, missing cement glands, abnormal development of the eye, and axial abnormalities (Creech Kraft and Juchau, 1995Go; Degitz et al., 2000Go; Pijnappel et al., 1993Go). When X. laevis were exposed to all-trans RA during a later stage of development (i.e., mid-limb bud stage), a concentration-dependent increase in the frequency and severity of hind limb malformations was observed (Degitz et al., 2000Go). Recently, Degitz et al.(2000)Go conducted experiments aimed at evaluating species differences in four North American ranid species and X. laevis. These studies compared the developmental toxicity of all-trans RA (as a model retinoid) in mid-blastula and/or limb bud stage X. laevis and several native North American Ranids. When tadpoles were exposed to all-trans RA during early- and mid-limb bud stages, significant species and stage-specific differences were observed ranging from no hind-limb abnormalities in several species to a concentration-dependent reduction and deletion of the hind-limbs in others. The underlying cause for the observed species difference may in part be attributed to differential uptake and metabolism of all-trans RA, although this possibility has not yet been explored.

Interpretation of retinoid dysmorphogenic effects can be enhanced through studies that examine the uptake and metabolism of the parent compound to potentially bioactive forms (Tzimas et al., 1994Go). Recently, a series of experiments were conducted that investigated the metabolism and dysmorphogenesis of all-trans RA and precursor retinoids in X. laevis during early stages of development (Creech Kraft and Juchau, 1995Go; Creech Kraft et al., 1995aGo,bGo). Results from these studies show embryos can metabolize all-trans RA to 13-cis RA, all-trans retinoyl ß-glucuronide (all-trans RAG), 4-oxo-all-trans RA, and 4-oxo-13-cis RA through a variety of isomerization, glucuronidation, and oxidation pathways. These retinoids and 9-cis RA, 5,6-epoxy RA, 4-hydroxy RA, and 3,4-didehydro RA have been identified as metabolites of all-trans RA in a number of mammalian and rodent studies (Barua et al., 1991Go; Blaner and Olson, 1994Go; Howard et al., 1989Go; Nau, 1993Go). Metabolites of all-trans RA identified by Creech Kraft and Juchau (1995)Go were further evaluated for their dysmorphogenic effects in X. laevis embryos. For example, 4-oxo-all-trans RA was found to be a potent dysmorphogen. This oxo-derivative was also shown to be a potent dysmorphogen in an earlier study with X. laevis embryos (Pijnappel et al., 1993Go). All-trans RAG was marginally teratogenic and likely represented a deactivation pathway for all-trans RA. Similarly, both 4-oxo-13-cis RA and 13-cis RA were less potent teratogens than their trans isomers and were also thought to be formed to inactivate all-trans RA. Both 4-oxo-all-trans RA and all-trans RA have been shown to specifically bind and activate RARs in X. laevis embryos (Pijnappel et al., 1993Go).

To further evaluate the species differences observed by Degitz et al.(2000)Go, the main objective of the experiments described herein was to examine the uptake and metabolism of all-trans RA in three native North American Ranid species: Rana sylvatica, Rana pipiens, and Rana clamitans. To our knowledge, this is the first investigation of all-trans RA uptake and metabolism in native anurans.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and reagents.
All-trans RA, 13-cis RA, 9-cis RA, all-trans retinol, and all-trans retinal were purchased from Sigma Chemical Company (St. Louis, MO). The oxo derivatives of RA (4-oxo-all-trans RA and 4-oxo-13-cis RA) were gifts from Hoffman-La Roche, Ltd. (Basel, Switzerland). Methanol (HPLC grade) and ethanol were purchased from Fisher Chemicals (Fair Lawn, NJ). 2-Propanol, suitable for liquid chromatography, was purchased from Burdick and Jackson Laboratories, Inc. (Muskegon, MI) and A.C.S. reagent grade ammonium acetate was obtained from Aldrich Chemical Company (Milwaukee, WI).

Organisms.
Newly-spawned R. pipiens were collected from a federal wetland near Roseau, MN, and R. clamitans were collected from Bass Lake in Bayfield County, WI, and returned to the laboratory within 48 h of spawning. Both sites were rural in nature and not adjacent to any obvious sources of contamination of agricultural runoff. R. sylvatica eggs were purchased from Carolina Biological (Burlington, NC) and maintained under the same conditions. The eggs were inspected and fertility/viability were determined to be greater than 95%. The organism were maintained in a continuous-flow of Lake Superior water at 22°C, under laboratory fluorescent light (16 h light:8 h dark) until the appropriate stage for testing was reached. Consequently, organisms were acclimated to Lake Superior water for at least a 2 week period before testing. Once hatched all organisms were fed a diet of trout starter/algae/tetrafin mixture and live brine shrimp three times daily (Ankley et al., 1998Go).

Tadpole exposures.
Rana pipiens, R. clamitans, and R. sylvatica were staged according to Gosner (1960)Go. Stage 28 R. clamitans and R. sylvatica and stage 26 R. pipiens were exposed to solvent control (0.05% ethanol), 250, 500, 750, 1000, or 1250 ng/ml of all-trans RA in Lake Superior water for 24 h under conditions of low light. Food was withheld during the 24 h exposure period. The all-trans RA concentration series used in our experiments is similar (e.g., 30 to 3000 ng all-trans RA/ml) to those used by others to examine amphibian developmental toxicity and/or metabolism of all-trans RA (Creech Kraft and Juchau, 1995Go; Kratke et al., 2000Go; Pijnappel et al., 1993Go). A stock solution of all-trans RA, in ethanol, was used to prepare the all-trans RA treatments, therefore the concentrations of ethanol in the all-trans RA treatments ranged from 0.01 to 0.05%. The R. pipiens and R. sylvatica treatment groups consisted of six replicates of 13 randomly selected organisms per 200 ml of test solution. The R. clamitans treatment group consisted of six replicates of 10 organisms per 200 ml of test solution. Fewer R. clamitans organisms were used in the all-trans RA treatment groups (i.e., 10 organisms) compared to the other two species (i.e., 13 organisms) due to the greater mass per organism ratio of the R. clamitans tadpoles. The density loading (i.e., organism mass per solution volume) for the R. pipiens, R. sylvatica, and R. clamitans experiments were 0.05, 0.13, and 0.24 g/ml, respectively. Aliquots of the exposure solutions (i.e., controls and all-trans RA treatments) were collected at 0 h and analyzed by HPLC to determine initial all-trans RA concentrations. Additional samples were collected at 4, 12, and 24 h to determine the rate of loss of all-trans RA from the exposure solutions. Tadpoles (i.e., 10 or 13 organisms) from the solvent control and five all-trans RA concentration levels were collected from two replicates per time point (i.e., replicates 1 and 2 sampled at 4 h, replicates 3 and 4 sampled at 12 h, and replicates 5 and 6 sampled at 24 h) to evaluate all-trans RA uptake and metabolism. Tadpoles were anesthetized with tricaine methanesulfonate (200 mg/l), frozen in liquid nitrogen and stored at -80°C prior to extraction.

Water analysis.
Water samples (0.5 ml) collected from the tadpole exposure solutions (i.e., controls and all-trans RA treatments) were placed into amber vials containing methanol (0.5 ml), mixed, and immediately analyzed for all-trans RA. Analyses were conducted using a Hewlett-Packard 1050 HPLC (Avondale, PA) equipped with a diode-array detector (wavelength, 354 nm) and a temperature-controlled (7°C) autosampler. An aliquot of the sample (300 µl injection) was injected directly into a Nucleosil C18 AB (250 x 4.6 mm) column (Alltech, Deerfield, IL) and all-trans RA was separated at a flow rate of 1 ml/min using a binary gradient program with mobile phase A, 40 mM ammonium acetate buffer (pH 7.3) and methanol (95:5, v/v) and mobile phase B, methanol. The gradient program was: 0 to 1.0 min at 95% A, followed by a linear gradient to 5% A in 30 min and held for 10 min. The HPLC method was similar to that reported by Creech Kraft and Juchau (1995)Go. Data were collected with Hewlett-Packard ChemStation software (version A.05.01) and all-trans RA concentrations determined using the external standard method of quantification with a seven-point linear calibration curve. Routine quality assurance analyses (matrix blanks, matrix spikes, and duplicate samples) were conducted with each sample set (e.g., one set of quality assurance samples were analyzed with every 12 exposure samples). No all-trans RA was detected in the blanks (n = 12). Recovery (mean ± SD) of all-trans RA in the spiked water samples from the R. sylvatica, R. pipiens, and R. clamitans exposures were 90 ± 3.7% (n = 4), 101 ± 1.0% (n = 3), and 96 ± 2.4% (n = 4), respectively. The agreement (mean ± SD) among duplicate samples from the R. sylvatica, R. pipiens, and R. clamitans exposures were 89 ± 8.6% (n = 2), 98 ± 2.8% (n = 4), and 85 ± 12.4% (n = 3), respectively. The analytical detection limit was 9 ng/ml.

Tissue analysis.
All sample manipulations were performed in a laboratory under amber lights to prevent photoisomerization and degradation of retinoids. Groups of organisms (i.e., 10 or 13 tadpoles) from each replicate were weighed (ca. 1–5 g), transferred to glass tubes containing ethanol (5 ml) and 2-propanol (10 ml), and placed on ice prior to extraction. The samples were homogenized using an Ultra-Turrax T25 tissue homogenizer (IKA works, Willington, NC) and quantitatively transferred using 2-propanol (5 ml) to polypropylene tubes. The homogenate was centrifuged at 3500 x g for 30 min at -5°C. The supernatant (i.e., extract) was removed and concentrated to 5 ml using a stream of purified nitrogen. To remove precipitated proteins, the extract was recentrifuged using the same conditions described above. The extract was transferred to a 10 ml volumetric flask and adjusted to volume with 2-propanol. An aliquot of the extract (300 µl) was placed into an amber vial and analyzed for all-trans RA by HPLC. An additional concentration step was needed for metabolite analysis; for this, an aliquot of the extract was concentrated fivefold (i.e., 2.5 ml to 0.5 ml), recentrifuged, transferred to an amber vial, and analyzed for retinoids by HPLC. Routine quality assurance samples (i.e., blanks and spikes) were extracted with each sample set. No all-trans RA was detected in the blanks (n = 9). Recoveries of all-trans RA in R. sylvatica, R. pipiens, and R. clamitans spiked samples were 85 ± 5.2% (n =3), 87 ± 2.6% (n = 3), and 81 ± 3.9% (n = 3), respectively.

Based upon the availability of analytical retinoid standards, the tissue extracts were analyzed for seven target retinoids by HPLC-DAD using conditions previously described for water analyses. These conditions allowed for separation of the target retinoids within 35 min. Figure 1AGo shows an HPLC separation of six of the seven target retinoids in an analytical standard mixture. All-trans retinal was not included in the mixture, but rather analyzed separately, due to some low level of impurity of all-trans retinol found in the all-trans retinal neat material. Samples were monitored at specific wavelengths for each analyte, which are similar to those reported by Furr et al.(1994)Go: 4-oxo-all-trans RA (360 nm), 4-oxo-13-cis RA (360 nm), 13-cis RA (354 nm), 9-cis RA (354 nm), all-trans RA (354 nm), all-trans retinol (325 nm), and all-trans retinal (380 nm). In addition during each sample run, spectral data were obtained for each chromatographic peak by scanning over the wavelength range from 190 nm to 600 nm. Compound identification was conducted by comparing chromatographic retention times and absorption spectra of the analytical reference standards to those in the tissue extracts. Retinoid concentrations were determined by the external standard method of quantification using linear calibration curves generated for each of the reference standards.



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FIG. 1. (A) HPLC chromatogram illustrating separation of a mixture of retinoid standards (500 ng retionid/ml). Peaks: 1, 4-oxo-all-trans RA; 2, 4-oxo-13-cis RA; 3, 13-cis RA; 4, 9-cis RA; 5, all-trans RA; 6, all-trans retinol. (B) Sample chromatogram of HPLC analysis of R. clamitans tadpoles exposed to all-trans-RA (750 ng/ml) for 4 h. Peaks 1–6 are the same as listed in (A). Peak 7, all-trans retinal, identified by elution with analytical standard.

 
Data analysis.
All-trans RA and associated metabolite concentrations are reported as mean ± SD (n = 2) at each time point and all-trans RA treatment level for the three species tested. The data were statistically analyzed using a two factorial design for each combination of species and chemical (Mason et al., 1989Go). The factors are all-trans RA treatment level and duration of the exposure. In general, the treatment level and duration were significant factors with limited interaction. Tukey-Kramer honestly significant difference (HSD) procedure was used to differentiate means of treatment levels associated with significant analyses of variance (Mason et al., 1989Go; Tukey, 1977Go). This method adjusts for post-hoc multiple comparisons of means. The two factor analyses of variance and mean separation procedure were performed with SYSTAT Version 9 (Wilkenson, 1999Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Toxicity of All-trans RA
No mortality was observed in R. sylvatica or R. clamitans tadpoles exposed to a wide range of all-trans RA concentrations (i.e., 250 to 1250 ng/ml) for 24 h. Initially, no mortality was observed in R. pipiens tadpoles exposed to all-trans RA; however by 24 h, 50 and 100% mortality occurred in R. pipiens tadpoles exposed to 1000 ng/ml and 1250 ng/ml of all-trans RA, respectively.

Aqueous All-trans RA Degradation
All-trans RA concentrations were monitored at each treatment level at 0, 4, 12, and 24 h for the R. sylvatica, R. pipiens, and R. clamitans experiments. All-trans RA concentration data from the R. pipiens exposure solutions has been previously reported (Degitz et al., 2000Go) and are similar to concentration data observed in the R. sylvatica and R. clamitans exposures (data not shown). Briefly, all-trans RA degraded rapidly in water, with approximately 70, 80, and 85% of the parent compound gone in 4 h in the R. pipiens, R. sylvatica, and R. clamitans exposures, respectively. No all-trans RA was detected by 24 h. It should be noted that, as concentrations of all-trans RA decreased in the static exposure solutions, two additional chromatographic peaks (monitored at wavelength 354 nm) were observed in the solutions at 4, 12, and 24 h (data not shown). The chromatographic retention times of these peaks, which were much earlier than all-trans RA (i.e., more polar), did not correspond with any of the seven target retinoid standards used in our analyses. Therefore, identification or quantification of these degradation products could not be determined. An evaluation of the potential biological activity of these degradation products was conducted by preparing additional all-trans RA solutions at concentrations of 750, 1000, and 1250 ng/ml and allowing the solutions to degrade for 24 h. Organisms were then placed in these solutions for 24 h under the same conditions used for the all-trans RA study. The degraded solutions were not toxic to the organisms and did not affect subsequent development (data not shown).

All-trans RA in Tadpoles
Rana sylvatica, R. pipiens, and R. clamitans tadpoles collected at 4, 12, and 24 h during the exposures were evaluated for whole body all-trans RA concentrations. As expected from the declining all-trans RA concentrations in the exposure solutions, tadpoles showed the greatest accumulation of all-trans RA at 4 h followed by decreasing concentrations at 12 and 24 h (Table 1Go). Rana pipiens had the highest concentrations of all-trans RA of the three species exposed. In general, at 4 and 12 h, all-trans RA concentrations in R. pipiens were approximately five times higher than concentrations in R. clamitans and 2.5 times higher than concentrations in R. sylvatica. Differences in all-trans RA concentrations observed among the species were most likely related to differences in organism density during the exposures. Specifically, densities were approximately 5 and 2.5 times higher in the R. clamitans and R. sylvatica exposures, respectively, compared to the densities of tadpoles in the R. pipiens exposure. By 24 h, all-trans RA concentrations in R. pipiens (i.e., at the lower treatments where no mortality was observed) were similar or slightly lower than concentrations in R. sylvatica or R. clamitans (Table 1Go).


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TABLE 1 Concentration (µg/g) of All-trans RA in R. sylvatica, R. pipiens, and R. clamitans during 24 h Exposure
 
Metabolism of All-trans RA
In addition to all-trans RA, several other chromatographic peaks were observed in the tissue extracts indicating metabolism by the tadpoles and the presence of endogenous retinoids (Fig. 1BGo). As previously mentioned, target analytes included: 4-oxo-all-trans RA, 4-oxo-13-cis RA, 13-cis RA, 9-cis RA, all-trans retinol, and all-trans retinal. Chemical structures and potential conversion pathways for these retinoids have been presented elsewhere (Creech Kraft and Juchau, 1995Go; Creech Kraft et al., 1995aGo).

All-trans RA was converted to 4-oxo-all-trans RA in R. sylvatica, R. pipiens, and R. clamitans tadpoles (Table 2Go). In R. sylvatica, the highest concentration of 4-oxo-all-trans RA was measured at 4 h followed by decreasing levels at 12 and 24 h, which is consistent with the trend observed for parent compound concentrations (Table 2Go). This trend was less apparent in R. pipiens and R. clamitans. In some cases, 4-oxo-all-trans RA concentrations were similar to or slightly higher at 12 h compared with 4 h levels, indicating a short term build-up or retention of 4-oxo-all-trans RA by these two species. By 24 h, concentrations had decreased dramatically. Overall, concentrations of 4-oxo-all-trans RA, at 4 and 12 h, were approximately 5 times lower in R. clamitans compared with concentrations in R. sylvatica and R. pipiens. However, when the data were normalized to all-trans RA concentrations (Table 3Go), the percentage of all-trans RA metabolized to 4-oxo-all-trans RA were similar in R. sylvatica (0.4%), R. pipiens (0.2%), and R. clamitans (0.2%) tadpoles. No 4-oxo-13-cis RA was detected in the three species.


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TABLE 2 Concentration (ng/g) of 4-oxo-all-trans RA in R. sylvatica, R. pipiens, and R. clamitans during 24 h Exposure
 

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TABLE 3 Percentage of All-trans RA Converted to Metabolites in R. sylvatica (RS), R. pipiens (RP), and R. clamitans (RC) Tadpoles
 
All-trans RA also was converted to 13-cis RA in all three species. Levels of 13-cis RA were similar across the species, with the exception of R. pipiens at the highest treatment level where mortality was observed (Table 4Go). In general, 13-cis RA concentrations were highest at 4 h and declined over the 24 h static exposure period. Percentages of all-trans RA metabolized to the 13-cis isomer were low (0.5 to 2%) in the three species (Table 3Go). The 9-cis RA isomer also was detected, but only in R. sylvatica and R. pipiens tadpoles (Table 5Go). Concentrations were fairly similar in the two species with levels highest at 4 h and decreasing near detection limits by 24 h. Parent compound conversion to 9-cis RA was 0.3% for both species (Table 3Go).


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TABLE 4 Concentration (ng/g) of 13-cis RA in R. sylvatica, R. pipiens, and R. clamitans during 24 h Exposure
 

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TABLE 5 Concentration (ng/g) of 9-cis RA in R. sylvatica, R. pipiens, and R. clamitans during 24 h Exposure
 
In all three species, all-trans retinol (vitamin A) was detected in control and all-trans RA treated organisms (Table 6Go). Vitamin A levels were similar in R. sylvatica and R. pipiens (i.e., at the lower treatments where no mortality was observed) and varied little over time and treatment. However, vitamin A concentrations in these tadpoles were nearly a factor of 10 lower when compared to R. clamitans from similar aqueous all-trans RA exposure levels. In addition, all-trans RA appeared to result in nearly a threefold increase in vitamin A levels in treated R. clamitans (i.e., organisms at higher treatment levels) compared with control organisms. On average, levels of vitamin A in R. sylvatica, R. pipiens, and R. clamitans were 60, 50, and 470 ng/g, respectively. The aldehyde form of vitamin A (all-trans retinal) was detected only in R. clamitans tadpoles and at much lower concentrations than all-trans retinol (Table 7Go). All-trans retinal was detected in both controls and treated organisms at a concentration of approximately 65 ng/g.


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TABLE 6 Concentration (ng/g) of all-trans Retinol in R. sylvatica, R. pipiens, and R. clamitans during 24 h Exposure
 

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TABLE 7 Concentration (ng/g) of All-trans Retinal in R. clamitans during 24 h Exposure
 
In addition to the retinoids identified in our study, several other chromatographic peaks were observed in the tissue extracts in all three species indicating more extensive all-trans RA metabolism (Fig. 1BGo). While the areas (i.e., detector response) of these additional chromatographic peaks were similar in magnitude to those of the identified metabolites (data not shown), identification of these analytes would require synthesis of retiniod standards and extensive analyses by liquid chromatography-mass spectrometry (LC-MS).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All-trans RA degraded rapidly in water during our 24 h static exposures. This rapid degradation was not surprising given the labile nature of the compound under conditions of light, oxygen, and temperature (Furr et al., 1994Go). Consistent with our study, all-trans RA degraded in an aqueous medium 2 to 4 h after exposure initiation in amphibian studies conducted by Creech Kraft and Juchau (1995)Go. In our study, tadpoles showed the greatest accumulation of all-trans RA at 4 h, which was expected based on declining all-trans RA concentrations in the static exposure solutions. Initially, it appeared that the accumulation of all-trans RA was different among the three species tested; however, upon closer inspection, these results could be explained by different organism densities used in the three experiments.

All-trans RA was converted to 4-oxo-all-trans RA and 13-cis RA in all three Ranid species, while 9-cis RA was detected in R. sylvatica and R. pipiens tadpoles. After normalizing metabolite concentrations to those of the parent compound, the percentage of all-trans RA converted to the identified metabolites was low and fairly similar (e.g., 0.1 to 2.5% conversion at 4 and 12 h) among the species. Consistent with our studies, 4-oxo-all trans RA and 13-cis RA have been identified in X. laevis embryos following exposure to all-trans RA (Creech Kraft and Juchau, 1995Go). However, differences in all-trans RA metabolism appear to exist between the native Ranid species and X. laevis. 4-oxo-13-cis RA, identified in X. laevis embryos (Creech Kraft and Juchau, 1995Go), was not detected in the native tadpoles we examined. Conversely, 9-cis RA was not identified as a metabolite of all-trans RA in X. laevis (Creech Kraft and Juchau, 1995Go) but was found in two of the Ranid species we tested. The 9-cis isomer of all-trans RA has been identified as a metabolite of all-trans RA in other studies (Blaner and Olson, 1994Go). Lastly, all-trans RAG was observed in X. laevis embryos following exposure to all-trans RA (Creech Kraft and Juchau, 1995Go) but could not be confirmed in our tadpoles due to the lack of an analytical standard.

We also examined the impact of all-trans RA on homeostasis of the endogenous retinoids all-trans retinal and all-trans retinol. Although endogenous all-trans retinol (vitamin A) was detected in control tadpoles from all three species, all-trans retinol levels were considerably higher (i.e., an order of magnitude) in R. clamitans tadpoles. Treatment with all-trans RA did not result in any obvious change in all-trans retinol concentrations in R. sylvatica or R. pipiens, however nearly a threefold increase was observed in R. clamitans. In addition, we detected only endogenous all-trans retinal in R. clamitans and the concentrations were not obviously modulated by all-trans RA. This differs from an amphibian study where an increase in endogenous all-trans retinal was observed in axolotl embryos following exposure to all-trans RA (Kratke et al., 2000Go). Our results indicate that exogenous all-trans RA differentially regulates vitamin A homeostasis in the three species examined. There is precedence for regulation of vitamin A homeostasis by exogenous all-trans RA. Rats treated with all-trans RA during gestation showed a decrease in retinol concentrations in both the maternal plasma and embryo (Collins et al., 1994Go).

Creech Kraft and Juchau (1995)Go found that X. laevis embryos could metabolize all-trans RA to 13-cis RA, all-trans RAG, 4-oxo-all-trans RA, and 4-oxo-13-cis RA. To delineate the potential role of these metabolites in dysmorphogenesis, they exposed embryos to the individual metabolites. They found that 4-oxo-all-trans RA was a potent dysmorphogen, with exposure at 2000 ng/ml producing similar effects (i.e., type and frequency of malformations) as compared with embryos exposed to all-trans RA at 1000 ng/ml. This oxo-derivative was also identified as a highly active metabolite of all-trans RA in an earlier X. laevis study where it was shown to modulate positional specification (Pijnappel et al., 1993Go). Creech Kraft and Juchau (1995)Go found all-trans RAG to be marginally teratogenic in X. laevis at the highest concentration tested (1000 ng/ml), and suggested that this represented a deactivation pathway for all-trans RA. Consistent with this, all-trans RAG has not been shown to bind to nuclear retinoid receptors (Blaner and Olson, 1994Go). Interestingly, in axolotl, all-trans RAG was only slightly less potent than all-trans RA with respect to alterations in embryonic development (Kratke et al., 2000Go). This effect however, was explained by deglucuronidation of all-trans RAG to all-trans RA. Also, in the X. laevis studies, both 4-oxo-13-cis RA and 13-cis RA (tested at 2000 ng/ml and 1000 ng/ml, respectively) were less potent teratogens than their trans isomers, and were also thought to be involved in the inactivation of all-trans RA (Creech Kraft and Juchau, 1995Go). Again, these compounds have not been found to bind to retinoid receptors (Mangelsdorf et al., 1994Go). Their teratogenicity was speculated to be caused by isomerization, through a reversible reaction, to the active trans forms, 4-oxo-all-trans RA and all-trans RA, which have been shown to specifically bind and activate RARs in X. laevis embryos (Pijnappel et al., 1993Go). Although 9-cis RA was not identified as a metabolite of all-trans RA in X. laevis, it was identified as a metabolite in our studies. 9-cis RA has been shown to be a highly potent retinoid (Creech Kraft et al., 1994Go), which binds with high affinity to both RARs and RXRs (Allenby et al., 1993Go; Tzimas et al., 1994Go). Overall, our results indicate that all-trans RA is rapidly metabolized in native Ranids species to potentially bioactive metabolites some of which are known ligands for RARs and RXRs.

Previously, we examined the effects of all-trans RA on hind-limb development in several Ranid species and X. laevis tadpoles (Degitz et al., 2000Go). Early- and mid-limb bud stage R. sylvatica, R. pipiens, R. clamitans, and X. laevis tadpoles were exposed to all-trans RA using the same conditions described in this article. However following the 24-h exposure, tadpoles were moved to clean water and maintained until forelimb emergence when they were assessed for abnormalities of the hind limbs. In R. sylvatica tadpoles exposed at stage 28 and X. laevis tadpoles exposed at stage 51 (X. laevis were staged according to Nieuwkoop and Faber, 1994Go), all-trans RA treatment resulted in a concentration-dependent increase in hind-limb malformations, with malformations consisting mainly of reductions in one or more of the limb segments (Degitz et al., 2000Go). However, all-trans RA was ineffective at inducing hind-limb abnormalities in stage 26 and 28 R. pipiens and stage 28 R. clamitans. In our current study, all-trans RA metabolites identified in R. sylvatica tadpoles included 4-oxo-all-trans RA, 13-cis RA, and 9-cis RA. However, these same metabolites were also detected in R. pipiens tadpoles, a species in which we could not produce hind-limb malformations. Further, 4, 12, and 24 h all-trans RA concentrations were highest in R. pipiens, indicating that parent concentrations in the tissue would not explain the previously described difference in malformations. Based on the results from the studies described here, we cannot conclude that differential metabolism accounts for the previously observed species differences in teratogenic response. Previously, we suggested that the apparent disparity in responses among the R. sylvatica, R. pipiens, and R. clamitans tadpoles may be explained by the differences in the rate at which the three species develop (Degitz et al., 2000Go). Over the course of those experiments, R. sylvatica developed nearly twice as fast as R. pipiens and R. clamitans. Thus, given the fact that all-trans RA rapidly degraded in aqueous solution, R. sylvatica tadpoles were exposed over a longer developmental period relative to the other two species. In support of such an explanation, we have shown that X. laevis and R. sylvatica, which have similar developmental rates, responded similarly (i.e., similar dose response) to all-trans RA exposure (Degitz et al., 2000Go). In the absence of additional data, this continues to be the most plausible explanation for differential responses among the species.

In conclusion, we only examined a small number of the potential metabolites that have been shown to form following exposure to all-trans RA; the possibility exists that species differences in other metabolites may be involved. The observation of a number of unidentified potential retinoid metabolites in amphibians exposed to all-trans RA is not unique to our studies. In other amphibian studies (Creech Kraft and Juchau, 1995Go), a number of unidentified retinoid metabolites were also observed in X. laevis embryos following treatment with exogenous retinoids, including all-trans RA. Collectively, these metabolism studies highlight the difficulties of conducting a comprehensive analysis of retinoid metabolism since standards for many of the potential metabolites are not available from commercial sources (i.e., require specialized synthesis). Additionally, identification of these metabolites would require analyses by LC-MS. Such an effort, however, could possibly identify species differences in metabolism that could account for differences in teratogenic responses. Nevertheless, our studies represent the most comprehensive analysis to date of all-trans RA metabolism in any native Ranid species.


    ACKNOWLEDGMENTS
 
We thank Richard Kolanczyk, Mitch Rosen, and two anonymous reviewers for their helpful comments on the manuscript. Also, we thank Greg Elonen for his valued technical assistance and James Balogh for conducting the statistical analyses.


    NOTES
 
The information in this document has been funded wholly by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contexts reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement/recommendation for use.

1 To whom correspondence should be addressed at U.S. EPA, Mid-Continent Ecology Division, Duluth, MN 55804-2595. Fax: (218) 529-5003. E-mail: kosian.pat{at}epa.gov. Back


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 RESULTS
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