In vitro degradation of hexanitrohexaazaisowurtzitane (CL-20) by cytosolic enzymes of Japanese quail and the rabbit

Hexanitrohexaazaisowurtzitane (CL‐20) is a polycyclic nitramine explosive and propellant, currently being considered as a potential replacement for existing cyclic nitramine explosives. Earlier studies have provided evidence suggestive of adverse liver effects in adult Coturnix spp. exposed to CL‐20, yet analysis of tissue samples (plasma, liver, brain, heart, or spleen) indicated that CL‐20 was not detectable in these treated animals. The present study was conducted to identify and purify the enzymes capable of CL‐20 biotransformation. Results indicate that the hepatic biotransformation of CL‐20 in vitro was inhibited by ethacrynic acid (93%) and by the glutathione (GSH) analogue S‐octylglutathione (80%), suggesting the involvement of glutathione‐S‐transferase (GST). Partially purified cytosolic α‐ and μ‐type GST (requiring presence of GSH as a cofactor) from quail and rabbit liver was capable of CL‐20 biotransformation. The degradation of CL‐20 (0.30 ± 0.05 and 0.40 ± 0.02 nmol/min/mg protein for quail and rabbit, respectively) was accompanied with the formation of nitrite and consumption of GSH. Using liquid chromatography/mass spectrometry, we detected two intermediates, that is, open‐ring, monodenitrated GSH–conjugated CL‐20 biotransformation product with the same deprotonated molecular mass ion at 699 Da, suggesting isomeric forms of the intermediate metabolites. Identity of the conjugated metabolites was confirmed by using ring‐labeled [15N]CL‐20 and the nitro group–labeled [15NO2]CL‐20. These data suggest that the in vitro biotransformation of CL‐20 by GST under the conditions tested may be a key initial step in the in vivo degradation of CL‐20 in the quail and resulted in the formation of more biologically reactive intermediates than the parent compound. These data will aid in our understanding of the biotransformation processes of CL‐20 in vivo.