Abstract
We examined the effects of acute isobutyl nitrite (ISBN) exposure on the activity of several hepatic enzymes. Two strains of adult male mice (Balb/c and C57BL/6) were exposed to 900 ppm ISBN or ambient air for 45 minutes. The enzyme activity of hepatic cytochrome P450 (CYP)-mediated deethylation, glutathione S-transferase (GST), and carboxylesterase (CBE) was monitored through the substrates 3-cyano-7-ethoxycoumarin (CEC), 1-chloro-2,4-dinitrobenzene, and p-nitrophenyl acetate, respectively. Acute ISBN exposure led to a significant reduction in hepatic CYP-mediated CEC deethylation, GST, and CBE activity in Balb/c mice (of 81.5%, 74.7%, and 25.2%, respectively, vs control mice, each at P<.05) when livers were harvested immediately after inhalant exposure. The corresponding decreases in C57BL/6 mice were smaller (with reductions of 21.8%, 18.8%, and 13.3%, respectively, each at P<.05). This enzyme activity, tested in C57BL/6 mice only, returned to control values after a 24-hour period of nonexposure. Follow-up mechanistic investigations using rat liver GST indicated that ISBN-mediated enzyme inactivation was not caused by its metabolites: inorganic nitrite ion (NO2 −) or nitric oxide. This inactivation could be prevented, but not reversed, by added glutathione, suggesting irreversible protein oxidation. Using different NO donors as comparative agents, we found that GST inactivation by ISBN was not associated with protein S-nitrosylation or disulfide formation, but with tyrosine nitration. Inhalant nitrite exposure, therefore, led to a significant reduction in hepatic enzyme activity in mice, possibly through tyrosine nitration of hepatic proteins. This effect raises the possibility of drug-drug metabolic interactions from inhalant nitrite abuse. However, determining the applicability of these findings to humans will require further study.
Similar content being viewed by others
References
Waugh M. Amyl nitrite as a sexual stimulant. BMJ. 1979;2:499.
Kielbasa W, Fung HL. Relationship between pharmacokinetics and hemodynamic effects of inhaled isobutyl nitrite in conscious rats. AAPS PharmSci. 2002;2:E11. serial online.
Tran DC, Yeh KC, Brazeau DA, Fung HL. Inhalant nitrite exposure alters mouse hepatic angiogenic gene expression. Biochem Biophys Res Commun. 2003;310:439–445.
Tran DC, Brazeau DA, Nickerson PA, Fung HL. Effects of repreated in vivo inhalant nitrite exposure on gene expression in mouse liver and lungs. Nitric Oxide. 2006;14:279–289.
Haverkos HW. Nitrite inhalant abuse and AIDS-related Kaposi's sarcoma. J Acquir Immune Defic Syndr. 1990;3:S47-S50.
Kielbasa W, Fung HL. Nitrite inhalation in rats elevates tissue NOS III expression and alters tyrosine nitration and phsophorylation. Biochem Biophys Res Commun. 2000;275:335–342.
Wong PS, Eiserich JP, Reddy S, Lopez CL, Cross CE, van der Vliet A. Inactivation of glutathione S-transferases by nitric oxide-derived oxidants: exploring a role for tyrosine nitration. Arch Biochem Biophys. 2001;394:216–228.
Ji Y, Neverova I, Van Eyk JE, Bennett BM. Nitration of tyrosine 92 mediates the activation of rat microsomal glutathione s-transferase by peroxynitrite. J Biol Chem. 2006;281:1986–1991.
Donato MT, Jimenez N, Castell JV, Gomez-Lechon MJ. Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab Dispos. 2004;32:699–706.
Coles BF, Kadlubar FF. Detoxification of electrophilic compounds by glutathione S-transferase catalysis: determinants of individual response to chemical carcinogens and chemotherapeutic drugs? Biofactors. 2003;17:115–130.
Bosron WF, Dean RA, Brzezinski MR, Pindel EV. Human liver cocaine carboxylesterases. NIDA Res Monogr. 1997;173:27–34.
O'Driscoll PT, McGough J, Hagan H, Thiede H, Critchlow C, Alexander ER. Predictors of accidental fatal drug overdose among a cohort of injection drug users. Am J Public Health. 2001;91:984–987.
Lee WI, Fung HL. Mechanism-based partial inactivation of glutathione S-transferases by nitroglycerin: tyrosine nitration vs sulfhydryl oxidation. Nitric Oxide. 2003;8:103–110.
Ponnappan U, Soderberg LS. Inflammatory macrophage nuclear factor-kappaB and proteasome activity are inhibited following exposure to inhaled isobutyl nitrite. J Leukoc Biol. 2001;69:639–644.
Kielbasa WB, Bauer JA, Fung HL. Analysis of isobutyl nitrite inhalant in rat and human blood: application for pharmacokinetic investigations. J Chromatogr B Biomed Sci Appl. 1999;734:83–89.
Emoto C, Yamazaki H, Yamasaki S, Shimada N, Nakajima M, Yokoi T. Characterization of cytochrome P450 enzymes involved in drug oxidations in mouse intestinal microsomes. Xenobiotica. 2000;30:943–953.
White IN. A continuous fluorometric assay for cytochrome P-450-dependent mixed function oxidases using 3-cyano-7-ethoxycoumarin. Anal Biochem. 1988;172:304–310.
Loscalzo J, Freedman J. Purification and characterization of human platelet glutathione-S- transferase. Blood. 1986;67:1595–1599.
Lee RP, Forkert PG. Inactivation of cytochrome P-450 (CYP2E1) and carboxylesterase (hydrolase A) enzymes by vinyl carbamate in murine pulmonary microsomes. Drug Metab Dispos. 1999;27:233–239.
Kielbasa W, Fung HL. Pharmacokinetics of a model organic nitrite inhalant and its alcohol metabolite in rats. Drug Metab Dispos. 2000;28:386–391.
Soderberg LS, Barnett JB. Inhalation exposure to isobutyl nitrite inhibits macrophage tumoricidal activity and modulates inducible nitric oxide. J Leukoc Biol. 1995;57:135–140.
Soderberg LS, Flick JT, Barnett JB. Leukopenia and altered hematopoietic activity in mice exposed to the abused inhalant, isobutyl nitrite. Exp Hematol. 1996;24:848–853.
Vuppugalla R, Mehvar R. Short-term inhibitory effects of nitric oxide on cytochrome P450-mediated drug metabolism: time dependency and reversibility profiles in isolated perfused rat livers. Drug Metab Dispos. 2004;32:1446–1454.
Vuppugalla R, Mehvar R. Hepatic disposition and effects of nitric oxide donors: rapid and concentration-dependent reduction in the cytochrome P450-mediated drug metabolism in isolated perfused rat livers. J Pharmacol Exp Ther. 2004;310:718–727.
Gunnarsdottir S, Elfarra AA. Glutathione-dependent metabolism of cis-3-(9H-purin-6-ylthio)acrylic acid to yield the chemotherapeutic drug 6-mercaptopurine: evidence for two distinct mechanisms in rats. J Pharmacol Exp Ther. 1999;290:950–957.
Belenghi B, Romero-Puertas MC, Vercammen D, et al. Metacaspase activity of Arabidopsis thaliana is regulated by S-nitrosylation of a critical cysteine residue. J Biol Chem. 2007;282:1352–1358.
Stamler JS, Singel DJ, Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Science. 1992;258:1898–1902
Tabrizi-Fard MA, Lee WI, Fung HL. Differential interactions of nitric oxide donors with rat oxyhemoglobin. Biochem Pharmacol. 1999;58:671–674.
Tseng CM, Tabrizi-Fard MA, Fung HL. Differential sensitivity among nitric oxide donors toward ODQ-mediated inhibition of vascular relaxation. J Pharmacol Exp Ther. 2000;292:737–742.
Irie Y, Saeki M, Kamisaki Y, Martin E, Murad F. Histone H1.2 is a substrate for denitrase, an activity that reduces nitrotyrosine immunoreactivity in proteins. Proc Natl Acad Sci USA. 2003;100:5634–5639.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published: July 27, 2007
Rights and permissions
About this article
Cite this article
Turowski, S.G., Jank, K.E. & Fung, HL. Inactivation of hepatic enzymes by inhalant nitrite—In vivo and in vitro studies. AAPS J 9, 32 (2007). https://doi.org/10.1208/aapsj0903032
Received:
Revised:
Accepted:
DOI: https://doi.org/10.1208/aapsj0903032