T8 AM Ecological Fate and Effects of Explosives and Related Compounds
Tuesday, 15 November 2005: 8:00 AM - 11:40 AM in 337-338

(BAR-1117-658447) Purification of Japanese quail and rabbit cytosolic glutathione S-transferases capable of biotransforming CL-20.

Bardai, G¹, Hawari, J¹, Spear, P², Grosse, S¹, Hoang, J¹, Sunahara, G¹, ¹ Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada² Research Center for Environmental Toxicology (TOXEN), University of Quebec at Montreal, Montreal, Quebec, Canada

ABSTRACT- Hexanitrohexaazaisowurtzitane (CL-20), a highly energetic polycylic nitramine is an emerging environmental contaminant. Our earlier studies showed that adult quail exposed to CL-20 led to increased liver weight and liver aspartate aminotransferase activities, and yet CL-20 was not detectable in quail brain, heart, spleen, or liver. The possibility that adult quail liver may biotransform CL-20 was considered. Liver homogenates were prepared from adult female Japanese quail (Coturnix coturnix japonica) prior to the CL-20 biotransformation assays and the glutathione S-transferase (GST) purification procedures. Data indicates that the disappearance of CL-20 using whole cytosol was inhibited in vitro by either ethacrynic acid or the glutathione (GSH) analogue, s-octylglutathione, and suggested the involvment of GST. Using size exclusion, and affinity chromatography, we purified and characterized a cytosolic GST from quail liver capable of biotransforming CL-20. The molecular masses of the purified proteins were about 28 and 27 kDA. Partial N-terminal sequence analysis showed both alpha and mu classes of GST, having 100% homology to chicken and quail GST described in the literature. For comparative purposes, we also purified a rabbit GST enzyme. With the requirement of GSH, purified enzyme preparations from quail and rabbit livers biotransformed CL-20 at the rates (SD) of 0.27 (0.05) and 0.13 (0.02) nmol/min/mg protein, respectively. Data suggest that the purified protein can biotransform CL-20, as evidenced by the concomitant formation of nitrite (NO₂^-) with CL-20 removal. These data suggest that GST purified from female quail liver is capable of biotransforming CL-20 in-vitro, and may explain the absence of CL-20 in liver in-vivo.

Key words: Japanese quail, rabbit, CL-20, GST