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

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.