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

Abstract

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.

Glyceryl trinitrate metabolism in the quail embryo by the glutathione S-transferases leads to a perturbation in redox status and embryotoxicity

“…The specific activities obtained for the whole quail embryo GSTs were comparable to previous reports (Bardai et al, 2006;Dai et al, 1996). That we found similar ratios of CDNB/GTN specific activity in crude extracts and purified GSTs from both the whole embryo (0.03) and embryonic eye (0.01) indicates that all of the important GTN biotransformation activity was purified from the crude extract.…”

“…5, lane A) or whole embryo (Fig. 5, lane B) purified using the GSH affinity column were within the 22-29 kDa range; this is consistent with the range of molecular masses reported for avian GSTs (24-30 kDa) (Bardai et al, 2006;Cameron et al, 1995;Hsieh et al, 1999). Thus, the enzymes purified from whole embryo and embryonic eye using the GSH affinity column are GST-type enzymes.…”

“…Previously, we demonstrated that alpha-and mu-type GSTs present in the subcellular fractions of the adult quail liver were capable of metabolizing other nitro containing compounds (Bardai et al, 2006); here, for the first time, we show that in the quail embryo both the alpha-and mu-types of GSTs are capable of GTN denitration. In the 24 h time course study, the amount of free nitrite present decreased dramatically, suggesting that the nitrite was irreversibly bound to proteins.…”

“…The 100 000 g supernatant (containing cytosol) was filtered through glass wool and used for further purification. All enzyme purification procedures, including fast protein liquid chromatography (FPLC ® ) (Amersham Pharmacia Biotech, Uppsala, Sweden), were performed as previously described (Bardai et al, 2006). Briefly, protein purification columns were pre-equilibrated in Buffer A (described above).…”

“…While GSTs are found in all species, seven classes of cytosolic GST are recognized in mammalian species and are designated alpha, mu, pi, sigma, theta, omega, and zeta based on their functional and structural properties (Hayes et al, 2005). Several hepatic glutathione S-transferases isozymes, including the alpha and mu forms, are capable of denitrating GTN into glyceryl dinitrate (GDN) and NO 2 - (Singhal et al, 1996); these GSTs are also present in avian species (Bardai et al, 2006;Dai et al, 1996;Hsieh et al, 1999). The ability of the GSTs to metabolize GTN is dependent upon the presence of glutathione ("-glutamyl-cysteinyl-glycine or GSH); the reaction is thought to proceed as follows: GTN + 2GSH !…”