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 alpha- and mu-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.
Bardai
Ghalib K
GK
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada.
Halasz
Annamaria
A
Sunahara
Geoffrey I
GI
Dodard
Sabine
S
Spear
Philip A
PA
Grosse
Stephan
S
Hoang
Johnston
J
Hawari
Jalal
J
D000595:Amino Acid Sequence; D000818:Animals; D001372:Aza Compounds / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002244:Carbon / Q000378:metabolism; D003370:Coturnix / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects* / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006571:Heterocyclic Compounds / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011817:Rabbits; D013997:Time Factors
C482139:2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane; D001372:Aza Compounds; D004791:Enzyme Inhibitors; D006571:Heterocyclic Compounds; D002244:Carbon; D005982:Glutathione Transferase