Phase-II conjugation ability for PAH metabolism in amphibians: characteristics and inter-species differences

The present study examines amphibian metabolic activity - particularly conjugation - by analysis of pyrene (a four ring, polycyclic aromatic hydrocarbon) metabolites using high-performance liquid chromatography (HPLC) with fluorescence detector (FD), a mass spectrometry detector (MS) system and kinetic analysis of conjugation enzymes. Six amphibian species were exposed to pyrene (dissolved in water): African claw frog (Xenopus laevis); Tago's brown frog (Rana tagoi); Montane brown frog (Rana ornativentris); Wrinkled frog (Rana rugosa); Japanese newt (Cynops pyrrhogaster); and Clouded salamander (Hynobius nebulosus); plus one fish species, medaka (Oryzias latipes); and a fresh water snail (Clithon retropictus), and the resultant metabolites were collected. Identification of pyrene metabolites by HPLC and ion-trap MS system indicated that medaka mainly excreted pyrene-1-glucuronide (PYOG), while pyrene-1-sulfate (PYOS) was the main metabolite in all amphibian species. Pyrene metabolites in amphibians were different from those in invertebrate fresh water snails. Inter-species differences were also observed in pyrene metabolism among amphibians. Metabolite analysis showed that frogs relied more strongly on sulfate conjugation than did Japanese newts and clouded salamanders. Furthermore, urodelan amphibians, newts and salamanders, excreted glucose conjugates of pyrene that were not detected in the anuran amphibians. Kinetic analysis of conjugation by hepatic microsomes and cytosols indicated that differences in excreted metabolites reflected differences in enzymatic activities. Furthermore, pyrenediol (PYDOH) glucoside sulfate was detected in the Japanese newt sample. This novel metabolite has not been reported previously to this report, in which we have identified unique characteristics of amphibians in phase II pyrene metabolism.