Dietary Retinoic Acid Induces Hindlimb and Eye Deformities in Xenopus laevis DEREK H. ALSOP,* ,† SCOTT B. BROWN, ‡ AND GLEN J. VAN DER KRAAK † Department of Zoology, University of Guelph, Guelph, Ontario, Canada N1G 2W1, and Aquatic Ecosystem Protection Research Branch, National Water Research Institute, Environment Canada, Burlington, Ontario, Canada L7R 4A6 This study investigated the effects of dietary retinoic acid (RA) on frog hindlimb development. Xenopus laevis (African clawed frog) tadpoles were fed a diet supplemented with 0, 1, 10, or 100 μg of RA/g of food for 2 or 5 d at different stages of metamorphosis. Hindlimb deformities were induced in the group fed 100 μg of RA/g of diet for 5 d. Exposures beginning at mid-hindlimb bud development induced bilaterally bent tibiafibula (bony triangles), while exposures later in hindlimb development induced deformities of the feet, including fusion of the 1st and 2nd clawed digits and reduced length of the 4th and 5th digits (due to reduced, missing, or misplaced phalanges). There were also cases of extra phalanges in the 5th digit. The eye was another target of RA exposure. In one experiment, 58% of the tadpoles fed 10 μg of RA/g had a smaller or absent right eye. Additionally, 11% of the tadpoles fed 100 μg of RA/g of diet developed a smaller or absent left eye. Waterborne heavy metals (Zn or Cu) modified RA effects on the hindlimb and eye. Co-exposure to metals and RA resulted in cases of unilateral bony triangles and reduced rates of smaller eyes. There were also cases of extra hindlimb digits in Zn-exposed animals. Dietary RA exposure in tadpoles can cause some deformities that differ from waterborne RA exposures in previous studies. RA also induced deformities that resemble those in affected wild frog populations (bony triangles), although the patterns of other deformities and missing segments (phalanges and metatarsals) are not similar to those documented in the wild. Introduction Retinoic acid (RA) regulates the expression of genes that control a diverse array of biological processes (1) including those linked to reproduction (2, 3) and embryonic develop- ment (1, 4). RA is specifically involved in limb development. For example, RA deficiency inhibits limb growth in the mouse (5) and zebrafish (Danio rerio)(6, 7) and limb regeneration in the axolotl (Ambystoma mexicanum)(8). Excess RA during amphibian metamorphosis also alters limb development, and studies with Xenopus laevis have shown that exposure to retinoids (RA or the RA precursor retinyl palmitate) induces hindlimb malformations including digit deformities, short- ening or bending of bones, and losses of limb segments (9- 11). There are recent reports of population declines (12, 13) and deformities in wild frogs (14-18). Several factors have been implicated as the causative agents responsible for the malformations including parasites (16, 19), UV light (20- 23), and environmental contaminants including insecticides (24) and agricultural runoff (14). Although multiple limbs or limb segments have received much of the attention, the most common deformities appear to be missing parts of the hindlimb (14, 17, 25). Few studies have considered if hindlimbs with missing segments are due to alterations in RA homeostasis. Animals may encounter increased internal RA due to environmental factors in two ways. First, compounds in the environment may mimic RA and bind to RA receptors. One study found compounds in pulp mill effluent that bind to the RA receptors and retinoid X receptors in fish (26). Second, exposure to toxicants may disrupt endogenous retinoid homeostasis. For instance, rats exposed to 2,3,7,8-tetrachlo- rodibenzo-p-dioxin (TCDD) have increased circulating RA levels (27). Whole body retinoid levels were increased in Rana temporaria tadpoles after maternal exposure to polychlo- rinated biphenyls (PCBs) (28). RA precursors such as retinal and retinol increase in the plasma of adult X. laevis after exposure to estrogen (29), although the effects of environ- mental estrogens on retinoid homeostasis have yet to be tested. This study tests the effects of RA in relation to timing, dose, and duration of exposure on metamorphosis in X. laevis. Heavy metals can also induce developmental abnormalities in X. laevis (30, 31); therefore, the effects of zinc (Zn) and copper (Cu) and their interactions with RA were also examined. We used a dietary RA exposure in these experi- ments for two reasons. First, this route of RA uptake has yet to be tested. It is unknown whether dietary exposures would induce similar responses to waterborne exposures, which were used in previous experiments (9, 11). Second, we chose a dietary exposure in an attempt to mimic the increase in circulating retinoid levels that has been reported in animals exposed to toxicants such as planar halogenated aromatic hydrocarbons. The patterns of RA-induced deformities will be compared to those reported in wild frog populations and laboratory studies. Materials and Methods Animals and Breeding. Fertilized eggs were obtained by mating X. laevis adults. To initiate breeding, three males and three females were injected in the dorsal lymph sac with 600 and 800 IU of human chorionic gonadotropin, respectively (Sigma-Aldrich Canada, Oakville, ON). Male/female pairs were then housed in one of three 30 L aquaria containing 10 L of aerated water. Amplexus, egg laying, and fertilization occurred within 12 h in a 20 °C, darkened room. Once mating was completed, adults were removed, and the fertilized eggs were left to develop. Unfertilized eggs and dead embryos were removed from the aquaria daily. Free-swimming tadpoles [stages 40-42 (32)] were transferred to 10 L green/ opaque plastic tanks, containing 5 L of aerated water (pH 8.2, Ca 2+ ) 105 mg/L, Mg 2+ ) 36 mg/L, Na + ) 24 mg/L, Cl - ) 52 mg/L, total hardness ) 410 mg of CaCO3/L, alkalinity ) 250 mg of CaCO3/L). Tanks had yellow plastic covers to reduce incoming light and help prevent RA breakdown. * Corresponding author telephone: (519)824-4120, ext. 56213; fax: (519)767-1656; e-mail: dalsop@uoguelph.ca. † University of Guelph. ‡ Environment Canada. Environ. Sci. Technol. 2004, 38, 6290-6299 6290 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 23, 2004 10.1021/es049765n CCC: $27.50  2004 American Chemical Society Published on Web 08/18/2004