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

Exposure of stage 9 quail (Coturnix coturnix japonica) embryos to glyceryl trinitrate (GTN) induces malformations that were associated in previous studies with an increase in protein nitration. Increased nitration suggests metabolism of GTN by the embryo. The goals of this study were to characterize the enzymes and co-factors required for GTN metabolism by quail embryos, and to determine the effects of in ovo treatment with N-acetyl cysteine (NAC), a precursor of glutathione (GSH), on GTN embryotoxicity. GTN treatment of quail embryos resulted in an increase in nitrite, a decrease in total GSH, and an increase in the ratio of NADP(+)/NADPH, indicating that redox balance may be compromised in exposed embryos. Glutathione S-transferases (GSTs; EC 2.5.1.18) purified from the whole embryo (K(m) 0.84 mM; V(max) 36 µM/min) and the embryonic eye (K(m) 0.20 mM; V(max) 30 µM/min) had GTN-metabolizing activity (1436 and 34 nmol/min/mg, respectively); the addition of ethacrynic acid, an inhibitor of GST activity, decreased GTN metabolism. Peptide sequencing of the GST isozymes indicated that alpha- or mu-type GSTs in the embryo and embryonic eye had GTN metabolizing activity. NAC co-treatment partially protected against the effects of GTN exposure. Thus, GTN denitration by quail embryo GSTs may represent a key initial step in the developmental toxicity of GTN.

Developmental toxicity of glyceryl trinitrate in quail embryos.

BACKGROUND: Although glyceryl trinitrate (GTN) is used extensively to treat angina and heart failure, little is known about its effects on the conceptus during organogenesis. The goal of these studies was to investigate the effects of GTN in a model organism, the quail (Coturnix coturnix japonica) embryo. METHODS: To identify the effects of GTN on quail embryo development, fertilized quail eggs (n = 10-12 eggs/group) were injected with GTN (0, 4.4, 44, or 440 µM) at Hamburger-Hamilton (HH) stage 0, 9, or 19 and examined 7 days later. Next, HH 9 embryos were injected with GTN (0, 0.88, 4.4, 8.8, 44, 88, and 440 µM, in 20 µL per egg) and examined 24-hours, 48-hours, or 72-hours postinjection. Finally, the developing eye on one side was exposed to GTN (44 µM) ex ovo and the tissue was probed for the presence of nitrated proteins. RESULTS: In ovo GTN exposure induced a dose-dependent increase in the number of malformed viable quail embryos with a maximal effect in HH 9 embryos. Microphthalmia, craniofacial, heart, and neural tube defects were elevated in GTN-exposed embryos. An increase in nitrated proteins was observed in the developing eye region of embryos exposed ex ovo to GTN. CONCLUSIONS: GTN treatment induced a variety of malformations in quail embryos. The presence of nitrated proteins suggests that organic nitrates, such as GTN, generate reactive nitrogen species. We hypothesize that GTN perturbations in the redox status of the embryo may underlie its developmental toxicity.

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.

Evaluation of youth preferences for rapid and innovative human immunodeficiency virus antibody tests.

OBJECTIVE: To determine youth preferences for Food and Drug Administration (FDA)-approved and investigational human immunodeficiency virus (HIV) antibody collection and testing methods before and after subjects learned of test result response times; to determine how influential test result response times are on participants' preferences. DESIGN: After health educators explained and demonstrated 6 different HIV antibody collection and testing strategies (3 saliva, 1 urine, and 2 fingerstick methods), participants completed a confidential survey about test method preference and tried the different testing methods. The participants had an opportunity to re-rank their test method preference after learning about each test's result response time. SETTING: Health education sessions in both clinical and community settings. PARTICIPANTS: Youths aged 12 to 24 years. RESULTS: An oral collection device with a rapid saliva test was the most highly preferred test method. The preference for this method and the rapid response test methods via fingerstick procedures improved significantly after subjects learned of the rapid result response time, while the other methods were given significantly lower preference rankings after subjects learned of the longer result response times. Shifts in preference rankings were not related to sex, age, ethnic group, experience with HIV testing, or practice of risk behaviors. CONCLUSIONS: Our research supports the use of noninvasive and rapid HIV testing methods with rapid response times for adolescents to assist in the early identification of HIV status, while offering HIV prevention opportunities and immediate linkage to care.