Evaluation of the Developmental and Reproductive Toxicity of Methoxychlor using an Anuran (Xenopus tropicalis) Chronic Exposure Model

Douglas J. Fort*,1, John H. Thomas*, Robert L. Rogers*, Andra Noll*, Clinton D. Spaulding*, Patrick D. Guiney{dagger} and John A. Weeks{dagger}

* Fort Environmental Laboratories, Stillwater, Oklahoma; {dagger} Product Safety & Environmental Assessment, SC Johnson & Son, Racine, Wisconsin

Received January 13, 2004; accepted July 13, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The chronic toxicity of methoxychlor to the South African clawed frog, Xenopus (Silurana) tropicalis, was evaluated using a life cycle approach. The chronic exposure period ranged from mid-cell blastula stage [NF (Nieuwkoop and Faber, 1994Go) stage 8] to 90 days of exposure, during which time the organisms generally completed metamorphosis and emerged as juvenile frogs. Methoxychlor concentrations ranged from 1 to 100 µg/l. Methoxychlor concentrations >10 µg/l caused delayed development. Organisms exposed to 10 µg/l methoxychlor for 30 days showed enlarged thyroid glands with follicular hyperplasia. No increase in mortality or external malformation was observed at any of the test concentrations during early embryo-larval development (NF stage 8 to NF stage 46; ca. 2 days exposure). A concentration-dependent increase in external malformations and internal abnormalities of the liver and gonads were noted after 90 days of exposure, however. Skewing of the sex ratio toward the female gender decreased ovary weight and number of oocytes, and increased oocyte immaturity and necrosis were noted at methoxychlor concentrations of 100 µg/l. Reductions in testis weight and sperm cell count were also detected at 100 µg/l methoxychlor. Results from these studies suggested that methoxychlor was capable of altering the rate of larval development, but did not adversely affect early embryo-larval development (2 days of exposure) as manifested in external malformations. Internal malformations, increases in the ratio of phenotypic females, were induced by chronic methoxychlor exposure. In addition, reproductive endpoints, most notably in the female specimens, were adversely affected by methoxychlor exposure. These studies add to the standardization and validation of a useful amphibian test methods capable of evaluating both reproductive and developmental effects of potential endocrine disrupting chemicals over a life cycle exposure.

Key Words: Xenopus tropicalis; life cycle assay; methoxychlor; developmental toxicity; reproductive toxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although partial life cycle test methods have been developed for amphibian species that evaluate early embryo-larval development and metamorphosis, no standardized and well-validated amphibian chronic or life cycle exposure assessment models capable of evaluating both reproductive and developmental effects of endocrine disrupting chemical (EDC) exposure are currently available. As a test species, Xenopus laevis has several primary disadvantages as a developmental model. First, X. laevis requires 1–2 years to reach sexual maturity, reducing the practicability of use in life cycle and multigenerational experiments (Hirsch et al., 2002Go). Second, cytogenetically, X. laevis is tetraploid, containing duplicated gene copies, many of which are nonfunctional. This cytogenetic organization complicates creating transgenic lines and analyzing gene regulation. X. (Silurana) tropicalis (Fig. 1), a close relative of X. laevis, shares virtually all of the advantages of X. laevis; but none of the disadvantages in an embryological model system. X. tropicalis features a much shorter life cycle (4–5 months). X. tropicalis utilizes a smaller diploid genome comprised of 20 chromosomes, with about 1.7 ± 109 base pairs, compared to X. laevis which has 36 chromosomes with ~3.1 ± 109 bp. X. tropicalis is also the only diploid species in the Xenopus genus, and a re-evaluation of morphological data and molecular evidence has conclusively shown that X. tropicalis is monophyletic with the rest of the Xenopus family. Unfortunately, little work in evaluating X. tropicalis as a toxicological tool has been performed to date. We recently evaluated the use of X. tropicalis as an alternative test organism for FETAX (Fort et al., in press a; Song et al., 2003Go) and found reasonable concordance between the two species.



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FIG. 1. Comparison of adult 1.5-year-old female Xenopus laevis (R) and Xenopus tropicalis (L).

 
Overall, amphibians represent a suitable model for monitoring reproductive performance (Fort and McLaughlin, 2003Go; Fort et al., 2001aGo), early embryo-larval development (ASTM, 1998Go; Bantle et al., 1998Go; Dumont et al., 1983Go; Fort et al., in press b, 2003Go; Fort and McLaughlin, 2003Go), and advanced development, including metamorphosis (Carr et al., 2003Go; Fort et al., 2000Go; 2001bGo; Fort and Stover, 1997Go) and sexual maturation (Hayes et al., 2002Go; Kloas et al., 1999Go; Pickford and Morris, 2003Go; Tavera-Mendosa et al., 2002aGo, 2002bGo). Collection of concurrent information on the effects of chemicals on the sensitivity of various developmental stages throughout the life cycle not only provides valuable hazard assessment information but also provides mechanistic clues concerning the modes of action of reproductive toxicants. We recently described the developmental and reproductive effects of the estrogenic organochlorine pesticide methoxychlor at various life stages of the South African clawed frog, Xenopus laevis, in an effort to determine stage-specific sensitivity (Fort et al., in press c). The battery of assays included a short-term (4 days) early embryo-larval assay (FETAX), a 30-day hind limb development assay, an 18-day metamorphic climax assay, and a 30-day adult reproduction assay. Results suggested that sensitivity to methoxychlor was most dramatic during the reproductive phase of the life cycle, that it was somewhat sensitive during metamorphosis, and that it was least sensitive during early embryo-larval development.

In an effort to evaluate the utility of X. tropicalis as a model system for evaluating life cycle effects in amphibians, the chronic toxicity of methoxychlor to the South African clawed frog, X. tropicalis, was evaluated and is presented in this report. These studies suggested that X. tropicalis could serve effectively as a tool for assessing both reproductive and developmental effects of potential toxicants, including EDCs.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Adult male and female X. tropicalis were acquired from Xenopus 1 (Dexter, MI). Chemicals and biochemicals used in the culture of X. tropicalis in this study were purchased from either the Sigma Chemical Company (St. Louis, MO) or Fisher Scientific (Houston, TX). Methoxychlor (99% purity) was purchased from Chem Service (West Chester, PA). Methoxychlor was dissolved in DMSO (99.5% purity, Sigma Chemical Co.) to prepare the appropriate stock solution. The final concentration of DMSO in each of the tests performed was held constant through the test series and did not exceed a final concentration of 0.5 % (v/v). Human chorionic gonadotropin (hCG), used to induce breeding in X. tropicalis, was dissolved in 0.9% (v/v) saline solution.

Animal care and breeding. Animal husbandry, breeding, and embryo collection were performed as described in American Society for Testing and Materials (ASTM) E 1439–98 (ASTM, 1998Go). Adult X. tropicalis were fed a Salmon chow (Rangen Company, Buhl, ID) diet and maintained in a continuous-flow culture system at a temperature of 26.5° ± 0.5°C. Breeding was induced using hCG. Adult male and female X. tropicalis organisms were administered a primer dose of 20 IU of hCG sc 48 h prior to mating. A primary dose of 100 IU was administered immediately prior to introduction to the breeding chambers. Embryos were collected, and the jelly coat was removed by exposing the embryos to a 2% (w/v) cysteine solution (pH 8.1) for 2 min.

Assay methodology. Experiments were performed in general accordance with ASTM E1439–98 (ASTM, 1998Go) with the following modifications. All tests were initiated at NF (Nieuwkoop and Faber, 1994Go) stage 8. Sets of 50 blastula-stage embryos were placed in each of two replicate 70-l aquaria (100 organisms per concentration) containing 1, 10, or 100 µg/l methoxychlor. Effects on external development, including stage of development (Bantle et al., 1998Go; Nieuwkoop and Faber, 1994Go), mortality, and external malformation, were evaluated at NF stage 46 (2 days) which represents FETAX-like exposure; and at 30-day intervals through 90 days. Under optimal laboratory conditions, which include daily feeding and water exchange at a temperature of 26.5° ± 0.5°C, X. tropicalis complete metamorphosis within 35–45 days post-fertilization (Fort et al., in press d). Experiments designed to evaluate reproductive fitness were conducted at the conclusion of the 90-day exposure. The methoxychlor stock solution was diluted in appropriate volumes of dechlorinated tap water to prepare the appropriate test dilutions. DMSO at a concentration of 0.5% (v/v) was maintained constant in all test concentrations. Because of the intensive water requirements, dechlorinated tap water was used in lieu of FETAX solution. Two separate tanks of 50 solvent-control embryos were exposed to 0.5% (v/v) DMSO in dechlorinated tap water. Treatment and control aquaria contained a total of 50 l of solution (organism density = 1 organism/l). The pH of the test solutions was maintained between 7.8 and 8.0 at a temperature of 26.5° ± 0.5°C. Dissolved oxygen and pH were monitored daily. Two separate experiments were performed.

All solutions were administered through a flow-through delivery system, set to provide a complete volume replacement every 24 h during the test period. An estimate of the range of NF stages found in a given replicate for each treatment was performed daily. This estimation was based on a random survey of 25 free-swimming organisms within each replicate. The objective of daily stage estimation was to provide a general assessment of developmental progress and determine if substantial variation in the rates of development existed within a given replicate throughout the study. This process of daily staging was performed to assess whether the organisms within a given replicate were developing consistently at similar rates, which is an indication of the quality and fitness of the test. Dead organisms were removed and the numbers recorded. At exposure day 30, day 60, and day 90, test organisms were anesthetized in 100 mg/l 3-aminobenzoic acid ethyl ester (MS-222) and individually staged in accordance with Nieuwkoop and Faber (1994)Go. Visual examination of the thyroid glands through the transparent larvae was performed on five organisms per replicate for each treatment and the control group. The organisms were then allowed to recover in dechlorinated tap water and returned to their respective treatment vessel for continued exposure. Tests were terminated after 90 days of exposure. At the completion of the 90-day exposure, juvenile organisms were anesthetized in MS-222 (1,000 mg/l) and necropsied; selective tissues were frozen at –20°C for residue analysis. For necropsy, eight randomly selected individuals of each gender from each treatment were specifically evaluated for total body weight, ovary weight, total number of oocytes, and oocyte stage distribution for females; and total body weight, testis weight, and sperm cell count for the male specimens. Ovaries and testes from 8 additional randomly selected test organisms (4 female and 4 male) were removed and frozen at –20°C for tissue residue analysis. Determination of gender and abnormalities associated with the gonads and liver were examined, aided by the use of a dissecting microscope. Although further histological examination of the thyroid glands was performed, only gross examination of the gonads and liver was conducted. Remaining specimens from each treatment were anesthetized in 1000 mg/l MS-222 and preserved in 3% (w/v) formalin, pH 7.0, as an archive.

Thyroid histopathology. Based on the results of the visual examination of thyroid gland size in the organisms exposured for 30 days, a separate set of organisms was exposed to 10 µg/l methoxychlor or dechlorinated tap water (control) for 30 days, fixed in Bouin's solution for 96 h, rinsed sequentially three times in 70% ethanol, and preserved in 10% neutral buffered formalin. After tissue trimming, in which the tails were severed, the tissues were placed in cassettes and sectioned (three 24 µm step section, caudal to rostral). Once thyroid tissue was verified in each slide preparation, the slides were stained with hematoxylin and eosin (H-E), examined by light microscopy, and photographed with a Nikon Koolpix 5000 digital camera.

Ovary morphology. Ovaries in Xenopus consist primarily of transparent connective tissue and oocytes. Oocyte morphometry was performed by gently teasing the connective tissue from the oocytes, allowing direct access for evaluating oocyte stage and health. Oocyte staging was performed in accordance with the criteria established by Dumont (1972)Go. Oocyte health was based on criteria established in Bantle et al. (1998)Go. In accordance with Dumont (1972)Go, oocyte differentiation is assessed on a scale of I to VI, with stage I representing a 50–300 µm transparent, immature oocyte, and stage VI representing a 1200–1300 µm fully mature oocyte capable of being fertilized. Vitellogenesis commences and yolk appears in the Xenopus oocyte at stage III (450–600 µm) (Hausen and Riebesell, 1991). Thus, the significance of distinguishing oocytes as being < or ≥ stage III is that the egg is either in a previtellogenic or a vitellogenic phase.

Sperm cell count. Prior to homogenization, the testes were grossly examined for external abnormalities. For the total sperm count, saline-merthiolate-Triton [SMT (1 ml/10 mg tissue) 0.9% (w/v) NaCl; 0.01% (w/v) merthiolate; 0.05% (v/v) Triton X-100] was used to maintain the tissues during homogenization (Fort et al., 2001aGo). Testes were placed in a clean scintillation vial with SMT, minced with scissors, and homogenized (Powergen 125, Fisher Scientific, Houston, TX) for 2 min. A sample was then placed into a hemacytometer and the spermatids were counted. At least three chambers were counted for each sample. If the totals were not within 10%, the samples were recounted.

Data analysis. Comparisons of developmental effects and reproductive fitness evaluations were performed by analysis of variance (ANOVA; Bonferroni t-test or Dunnett's test, p < 0.05). Methoxychlor concentrations were determined by gas chromatography by means of electron-capture detection (GC-ECD). Practical quantitation limits (method detection limit * dilution factor) for methoxychlor were 0.1 µg/l and 50.0 ng/g, for water samples and tissue samples, respectively. Stock test solutions were measured weekly throughout the exposure period. Tissue samples from the reproductive studies including ovary, testis, and carcass (whole body without ovaries or testes) were collected from each trial and frozen until analysis. Whole ovary and testis samples from four organisms and carcass samples from each of the eight organisms were composed into one sample for each concentration per trial.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Control
Developmental assessment. Control treatments [0.5% (v/v) DMSO] developed at a rate consistent with that found previously by Fort et al. (in press a, d) and Hirsch et al. (2002)Go. Daily stage estimation indicated that of the 25 organisms surveyed, control organisms ranged no more than 2 NF stages within or between each replicate. By 30 days, control organisms reached NF stage 58–60 (Tables 1 and 2), and at least 80% metamorphosed by 40 days in culture. No external malformation was noted during early embryo-larval development between NF stage 8 and NF stage 46 (2 days of exposure consistent with FETAX). All living organisms in the control treatment groups metamorphosed by the subsequent evaluation period, 60 days. The frequencies of control mortality and external malformation were ≤18% and ≤11%, respectively. Control sex ratios were 49:51% and 50:50% female:male in trials 1 and 2, respectively. No signs of internal abnormalities were found in either trial.


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TABLE 1 Trial 1—Effects of Chronic Methoxychlor on Xenopus tropicalis Development1

 

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TABLE 2 Trial 2—Effects of Chronic Methoxychlor on Xenopus tropicalis Development1

 


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FIG. 2. Effect of 30-day methoxychlor exposure on thyroid glands of X. tropicalis (NF stage 58). A–Top, left: Control with arrows denoting paired thyroid glands and normal follicular development (H-E stained, 2.5x). B–Top, right: 10 µg/l methoxychlor exposed prometamorphic larvae, with arrows denoting paired thyroid glands (H-E stained, 2.5x). C–Bottom, left: Control, with top arrow denoting normal cuboidal follicular epithelium, middle arrow denoting normal homogeneous colloid, and bottom arrow noting follicular pigment (H-E stained, 10x). D–Bottom, right: 10 µg/l methoxychlor exposed prometamorphic larvae, with top arrow denoting follicular hyperplasia, middle arrow denoting homogeneous colloid, and bottom arrow noting follicular pigment (H-E stained, 10x).

 
Reproductive assessment. Control female specimens were found to be reproductively competent, with viable ovaries containing maturing oocytes and low incidences of necrosis (14.1 ± 2.8 and 10.5 ± 2.8 in trials 1 and 2, respectively; Tables 3 and 4). Control male specimens were also reproductively intact, with sperm cell counts (Tables 3 and 4) reasonably consistent with the level found in X. laevis (Fort et al., 2001aGo).


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TABLE 3 Trial 1—Summary of the Effects of Methoxychlor on Reproductive Endpoints in Juvenile X. tropicalis

 

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TABLE 4 Trial 2—Summary of the Effects of Methoxychlor on Reproductive Endpoints in Juvenile X. tropicalis

 
Developmental Effects—Methoxychlor Exposure
The effect of methoxychlor exposure on X. tropicalis development is expressed in Tables 1 and 2. Daily stage estimation for the various methoxychlor treatments, based on the 25 organisms surveyed per replicate of each treatment, indicated that daily stages ranged no more than 2 NF stages within or between replicates for the 1 µg/l methoxychlor treatment. More variation in daily stage distribution was noted in the organisms exposed to 10 or 100 µg/l methoxychlor. In either case, this range in NF stages became more dramatic with increased exposure length. Daily NF stage estimates in organisms exposed to 10 µg/l or 100 µg/l methoxychlor showed a range of no greater than 4 NF stages within or between each respective replicate. Variability estimates were reasonably consistent with the more robust staging data collected at each 30-day exposure interval. In both trials, slight developmental delay, based on mean stage obtained at the 30-day, 60-day, and 90-day intervals, was observed in each treatment, but delay was more pronounced in the 10 and 100 µg/l concentrations. At least 80% of the organisms exposed to the 1 µg/l metamorphosed by 45 days in culture, and by 65 days for the organisms exposed to 10 µg/l methoxychlor. None of the organisms exposed to 100 µg/l methoxychlor metamorphosed by the conclusion of the test at 90 days. This pronounced delay in development in the 100 µg/l methoxychlor organisms was observed progressively throughout the exposure at days 30, 60, and 90 in both trials.

Throughout the exposure period, the frequencies of mortality were not appreciably greater than observed in the controls (Tables 1 and 2). As noted with the controls, no signs of early external embryo-larval malformation was observed in any of the methoxychlor treatments after 2 days of exposure (NF stages 8–46; FETAX). At exposure day 30, a slightly increased frequency of external abnormal development was detected in organisms from the 10 µg/l and 100 µg/l methoxychlor treatments. This trend was more marked with increased exposure time, such that the frequency of external malformations identified in the 100 µg/l methoxychlor exposure treatments after 90 days of exposure was greater than the control (ANOVA, Bonferroni t-test, p < 0.05). External malformations observed in the 100 µg/l treatment included abnormal development of the gut and flexure of the tail resulting from slight disorganization of the mytotomes. These malformations were of moderate severity. A decrease in ovary size and increase in oocyte immaturity and necrosis, a unilateral decrease in testicular size and increase in testicular anomalies, mottling and necrosis of the liver which included the presence of tumors, and enlarged thyroid glands demonstrating follicular hyperplasia were noted compared to control treatments. These internal abnormalities were found in organisms from both the 10 µg/l and 100 µg/l methoxychlor treatments. The effects of methoxychlor exposure on thyroid, gonad, and liver development are illustrated in Figures 2Go4, respectively.



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FIG. 3. Effect of chronic (90-day) methoxychlor exposure on X. tropicalis gonadal development. A–Top, left: Control ovary. B–Top, right: Ovary from 100 µg/l methoxychlor-treated organism. Note reduced ovary size and immaturity and necrosis of oocytes. C–Bottom, left: Control testes. D–Bottom, right: Testes from 100 µg/l methoxychlor-treated X. tropicalis. Note reduced size and misshapen morphology of right testis resulting from atrophy. Also, note the unilateral nature of response.

 


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FIG. 4. Effect of chronic (90-day) methoxychlor exposure on liver development in X. tropicalis. A–Top: Control liver. B–Bottom, left: Liver from organism exposed to 100 µg/l methoxychlor. Note presence of tumors. C–Bottom, right: Liver from organism exposed to 100 µg/l methoxychlor. Note mottling and regional lesions.

 
Sex ratios in the 1 µg/l and 10 µg/l treatments were reasonably consistent with the control (Tables 1 and 2). However, the 100 µg/l treatment produced 78.2% and 89.2% female X. tropicalis in trials1 and 2, respectively, which was significantly greater than the control (ANOVA, Bonferroni t-test, p < 0.05).

Reproductive Fitness
Female. The effects of chronic methoxychlor exposure on reproductive status in female X. tropicalis are presented in Tables 3 and 4. A significant reduction in ovary weight, but not total body weight, compared to the control, was observed in the 100 µg/l methoxychlor treatment (ANOVA, Dunnett's test, p < 0.05). Reduction in the total number of eggs present in the ovaries, an increased presence of immature oocytes (< stage III), and increased presence of necrotic eggs was found in female specimens from the 100 µg/l methoxychlor treatment. Although these reproductive endpoint changes were found at 100 µg/l, the other treatments were not significantly different from the control (ANOVA, Dunnett's test, p < 0.05).

Male. The effects of chronic methoxychlor exposure on male reproductive status in X. tropicalis are presented in Tables 3 and 4. A significant reduction in whole body and testis weight compared to the control was observed in the 100 µg/l methoxychlor treatment (ANOVA, Dunnett's test, p < 0.05). A reduction in sperm cell count was detected in organisms from the 100 µg/l methoxychlor treatments (ANOVA, Dunnett's test, p < 0.05).

Tissue Residues
Accumulation of methoxychlor was noted in the carcass, but not to the same extent found in the ovary or testis samples (Table 5). This trend was consistent in both trials.


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TABLE 5 Methoxychlor Accumulation in Juvenile X. tropicalis Tissues1

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Results from the present study strongly indicated that chronic exposure to methoxychlor: (1) delayed development (including slowing the rate of metamorphosis), (2) had a slight effect on the normalcy of larval development, (3) induced follicular hyperplasia of the thyroid glands in prometamorphic larvae, (4) caused a skewed sex ratio toward the female gender, (5) and induced gonad and liver abnormalities in juvenile specimens. A few isolated cases of inter-sex gonad development were also found in this study. Chronic methoxychlor exposure also impaired both female and male reproductive fitness. In females, a concentration-dependent decrease in ovary weight, total number of oocytes present in the ovaries, oocyte maturity, and an increase in oocyte necrosis was observed. In males, less of a concentration-dependent response was observed. However, in the 100 µg/l methoxychlor exposure treatment, decreased testis weight and sperm cell counts were noted. Methoxychlor was found to bioaccumulate in the carcasses, but more dramatically accumulated in the gonads of X. tropicalis.

The results obtained from the present study were reasonably similar to those obtained from studies with X. laevis (Fort et al., in press c). Fort and co-workers (in press c) evaluated different stages of the life cycle of X. laevis in order to establish which stages demonstrated specific sensitivity to methoxychlor. Exposure scenarios targeted early embryo-larval development (NF stage 8–46 (FETAX)), 30-days hind limb development (NF stage 8–54), 18-days metamorphic climax (NF stage 58–66), and a reproductive evaluation of adult female and male organisms. Results from those studies indicated that the early developmental phase study was remarkably insensitive. However, delayed development, including differentiation of hind limb digits and inhibition of metamorphic climax, was observed. Delayed metamorphic climax was characterized by a slowing in the rate of tail resorption and a delay and dampening T3 surge characteristic of the onset of metamorphic climax. In addition, an effect on reproductive fitness in both adult female and male X. laevis associated with methoxychlor exposure was found. In female X. laevis, decreased ovary weight, decreased number of total oocytes within the ovary, increased numbers of immature oocytes (< stage III, Dumont, 1972Go) were noted. Decreased testis weight and sperm cell count were found in adult male X. laevis exposed to methoxychlor. In addition to these findings, pathological effects on the thyroid gland, characterized by follicular hyperplasia and abnormal testicular development, were also found in methoxychlor-exposed frogs. Based on crossover breeding studies, the reproductive effects induced by methoxychlor exposure in adult X. laevis females cited by Fort et al. (in press c), resulted in decreased reproductive outcome as measured by fertilization and viability of the progeny. The reduced reproductive fitness found in methoxychlor-exposed males did not necessarily translate into reduced reproductive performance as noted in the female specimens, however. Methoxychlor was found to accumulate in the gonads of both female and male X. laevis. In comparison, methoxychlor did not induce appreciable external malformation during early embryo-larval development (through NF stage 46) in either study, but it did cause developmental delay and potential disruption of thyroid axis activity. Reproductive toxicity in both female and male specimens was also found in both studies. Both studies also showed the propensity of methoxychlor to accumulate in the reproductive tissues, although methoxychlor accumulated to a greater extent in the testis in X. tropicalis than in X. laevis. The present study with X. tropicalis found that methoxychlor was capable of inducing abnormal development of the gonads and pathology associated with the liver. The greatest difference in results between the two studies appeared to be in the potency of methoxychlor to induce chronic toxicity. In the case of reproductive toxicity, many of the adverse effects found in female X. laevis were detected at concentrations <10 µg/l methoxychlor, whereas the reproductive effects in X. tropicalis were primary observed at concentrations >10 µg/l methoxychlor. In terms of developmental effects, however, X. tropicalis appeared to be more sensitive. These differences in sensitivity may have been the result of many factors, most notably, exposure method and duration, as well as differences in species sensitivity. Overall, the results were reasonably consistent between the studies, which validated both the responses and the test methods used.

As previously indicated, X. tropicalis offers several advantages over X. laevis as a toxicological test tool. One of the most notable attributes in selecting an anuran species that could be effectively used for chronic studies (including multigenerational experimental designs) is the shorter time required to reach sexual maturity. Because sexual maturity in X. laevis requires 1–2 years, but only 4–5 months in X. tropicalis, the latter represents a more suitable choice of test species for life cycle–type tests. Although effects of methoxychlor on the complete life cycle of X. tropicalis are not reported in this study, subsequent studies in our laboratory evaluating full life cycle and multigenerational effects of EDCs (data not reported) are now demonstrating the potential utility of this species. Results from the present study warrant additional study to continue development of chronic and life cycle protocols. This study supports the continued work in the use of X. tropicalis in the field of environmental toxicology.

A number of studies have evaluated the toxicological effects of methoxychlor on reproduction and early development in amphibians (Bevan et al., 2003Go; Eroschenko et al., 2002Go; Hall and Swineford, 1979Go; Ingerman et al., 1999Go, 2002Go; Pickford and Morris, 1999Go, 2003Go; Verrell, 2000Go). Of these studies, only one investigation focused on the effects of methoxychlor on early embryo-larval development (Bevan et al., 2003Go). Although these investigators found that other environmental estrogens, such as, nonylphenol and octylphenol, as well as the natural estrogen 17ß-estradiol, were capable of inducing gross abnormalities at NF stage 37 (48 h of development) at µM concentration ranges, methoxychlor did not induce gross malformation. Bevan et al. (2003)Go, however, did find that methoxychlor and the other common environmental estrogens altered neural crest melanocyte differentiation. Because expression of the early neural crest marker Xslug, a factor that regulates both the induction and the movement of neural crest cells early in development, was not altered by methoxychlor exposure, these investigators suggested that the effect of methoxychlor may occur near the end of melanocyte differentiation. Most of the remaining studies of methoxychlor effects in amphibians focus on the effects of methoxychlor on reproductive fitness (Pickford and Morris, 1999Go, 2003Go) and ecological and behavioral effects (Eroschenko et al., 2002Go; Hall and Swineford, 1979Go; Ingermann et al., 2002Go; Verrell, 2000Go). Pickford and Morris (1999)Go demonstrated that methoxychlor was a potent inhibitor of oocyte maturation in X. laevis when oocytes cultured in vitro were exposed to varying concentrations of methoxychlor, as indicated by the occurrence of germinal vesicle breakdown (GVBD). These investigators have subsequently shown that gonadotropin-induced ovulation was altered in X. laevis exposed to environmentally relevant concentrations of methoxychlor (Pickford and Morris, 2003Go). In addition, skews in plasma sex steroid ratios after oviposition and decreased in vitro progesterone production in ovarian explants were also noted to occur after methoxychlor exposure. These studies further demonstrate the capacity of methoxychlor to negatively affect reproductive fitness and have potential population-level implications.

Unfortunately, few if any, studies with methoxychlor have demonstrated the effects of methoxychlor on advanced development, including metamorphosis in amphibians. Cheek et al. (1999)Go demonstrated that the pre-emergent herbicide acetochlor accelerated T3-induced metamorphosis in R. pipiens, apparently by positively interacting with T3 via a non-thyroid hormone-related mechanism. Moreover, little toxicological study has been performed with X. tropicalis (Fort et al., in press a, b; Song et al., 2003Go). Most of the work performed in X. tropicalis has been in the fields of developmental biology with a molecular emphasis (Beck and Slack, 2001Go; Blackshear et al., 2001Go; Carruthers et al., 2003Go; Chae et al., 2002Go; Offield et al., 2000Go). The effect of methoxychlor on advanced development, endocrine activity, reproduction, and thyroid function in mammals has been reasonably well studied (Amstislavsky et al., 2003Go; Borgeest et al., 2002Go; Golub et al., 2003Go; Gray et al., 1989Go; Johnson et al., 2002Go; Lafuenta et al., 2003Go; Latchoumycandane and Mathur, 2002Go; Wade et al., 2002Go; Zhou et al., 1995Go). Golub et al. (2003)Go found that female rhesus monkeys administered 25 and 50 mg/kg/day methoxychlor during the peripubertal period showed reduced growth and suppressed menses. Exposure also led to premature emergence of secondary sexual characteristics, caused an increase in the formation of ovarian cysts; treated females also demonstrated shorter follicular phases. Alterations in ovarian cycles in X. tropicalis as the result of methoxychlor exposure were also noted. Methoxychlor has been shown to alter episodic prolactin release in adult male Sprague-Dawley rats by sensitizing the pituitary to thyrotropin releasing hormone (TRH) (Lafuenta et al., 2003Go). Amstislavksy et al. (2003)Go found that administration of 16.5 mg/kg methoxychlor to pregnant mice on day 2–4 did not interfere with tubal embryo transfer or induce malformations. However, these investigators did suppress embryo development, as was found in the present study in X. tropicalis, and increased the frequency of nuclear fragmentation and the formation of micronuclei. Borgeest et al. (2002)Go determined that administration of methoxychlor ip for 10–20 days at a dose of 32 mg/kg/day caused an increase in the ovarian surface epithelial height and increased antral follicular atresia. Adverse effects of methoxychlor exposure on the testis have also been demonstrated by Johnson et al. (2002)Go and Latchoumycandane and Mathur (2002)Go. Johnson et al. (2002)Go found that perinatal rat dams gavaged with 5–150 mg/kg/day for 1 week prior to and 1 week after birth, followed by a subsequent exposure of male rats to methoxychlor from postnatal days 7 to 42 caused reduced testicular size and epididymal spermatozoa, and the number of Sertoli cells. Latchoumycandane and Mathur (2002)Go found similar morphological effects in male rats, as well as signs of increased oxidative stress in the testis.

Gray et al. (1989)Go, Wade et al. (2002)Go, and Zhou et al. (1995)Go each found that methoxychlor had a thyroid toxic effect. Gray et al. (1989)Go found that, in addition to endocrine disturbance at the gonadal level, pituitary levels of prolactin, follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH) were altered. Serum TSH was reduced by at least 50% compared to controls at 100 mg/kg/day, whereas pituitary levels increased. In the present study, thyroid follicular hyperplasia was found in X. tropicalis treated with methoxychlor.


    ACKNOWLEDGMENTS
 
The authors thank Ms. Michelle Gilliland for her help in preparing this manuscript. This work was supported by funding from S.C. Johnson & Son, the U.S. Environmental Protection Agency (USEPA) under contract no. 68-D-03–024, and the Oklahoma Center for the Advancement of Science & Technology (OCAST) under grant no. AR012–022. Research was conducted in compliance with the Animal Welfare Act and other federal statues and regulations relating to animals and experiments involving animals and adhered to principles stated in the Guide for Care and Use of Laboratory Animals (NRC 1996).


    NOTES
 

1 To whom all correspondence should be addressed at Fort Environmental Laboratories, 1414 South Sangre Road, Stillwater, Oklahoma 74074. Fax: (405) 377–3388. E-mail: djfort{at}fortlabs.com.


    REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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