Strong Protein Adduct Trapping Accompanies Abolition of Acrolein-Mediated Hepatotoxicity by Hydralazine in Mice

Abstract

Acrolein is a highly reactive ␣,␤-unsaturated aldehyde that readily alkylates nucleophilic centers in cell macromolecules. Typically, such reactions proceed via Michael addition chemistry, forming adducts that retain an electrophilic carbonyl group. Since these species participate in secondary deleterious reactions, we hypothesize that inactivation of carbonyl adducts may attenuate acrolein toxicity. Indeed, we recently established that the nucleophilic antihypertensive drug hydralazine readily "traps" acrolein adducts in cell proteins and strongly suppresses acrolein-mediated toxicity in isolated hepatocytes. This work sought to determine whether hydralazine prevents the in vivo hepatotoxicity of the acrolein precursor allyl alcohol in whole mice and whether adduct trapping accompanies any such hepatoprotection. Mice received allyl alcohol alone or in conjunction with several doses of hydralazine. Four hours later, mice were sacrificed to allow for the determination of liver enzymes in plasma as markers of hepatic injury, whereas livers were assessed for glutathione and hydralazine-stabilized protein adducts. Hydralazine afforded strong, dose-dependent protection against the increases in plasma marker enzymes but not the hepatic glutathione depletion produced by allyl alcohol. Western blotting revealed intense, dose-dependent adduct trapping by hydralazine in numerous liver proteins over a broad 26-to 200-kDA mass range. In keeping with these findings, immunohistochemical analysis of liver slices indicated diffuse, extranuclear adduct trapping by hydralazine that was uniformly distributed across the liver lobule, with partial localization in parenchymal cell membranes. These findings concur with our hypothesis that hydralazine readily inactivates reactive carbonyl-retaining protein adducts formed by acrolein, thereby preventing secondary reactions that trigger cellular death.

Hydralazine Inhibits Rapid Acrolein-Induced Protein Oligomerization: Role of Aldehyde Scavenging and Adduct Trapping in Cross-Link Blocking and Cytoprotection

“…We identified the antihypertensive hydralazine, among the strongest nucleophiles in clinical use, as a powerful inhibitor of acrolein toxicity (Lalich and Paik, 1975;Burcham et al, 2002). Although its protective efficacy seemed to be related to its acrolein-scavenging properties in cell-free systems (Kaminskas et al, 2004b), we found that hydralazine also reacts extensively with adducts formed during protein modification by acrolein (Burcham et al, 2004;Kaminskas et al, 2004b). The present work breaks new ground by establishing that the latter adduct-trapping reactivity is more closely related to hydralazine cytoprotection than the direct acrolein-scavenging reaction (Fig.…”

“…Using rabbit antiserum against drug-labeled adducts, we also detected strong adduct-trapping in a wide range of cell proteins from mouse hepatocytes treated briefly with allyl alcohol and cytoprotective concentrations of hydralazine (e.g., 2 to 50 M; see Burcham et al, 2004). Moreover, intense protein adducttrapping occurred in the livers of allyl alcohol-intoxicated mice that received hepatoprotective doses of hydralazine (Kaminskas et al, 2004b).…”

Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism

“…The reaction was started by adding 15 mL of 10 mmol/L NADPH. AR activity was measured at 340 nm at 25 C for 5 min.…”

“…24 Acrolein can cause hepatotoxicity in vitro and in vivo. [25][26][27][28] However, the mechanisms involved in acrolein-induced hepatocellular toxicity are not completely understood.…”

Chaperone Heat Shock Protein 90 Mobilization and Hydralazine Cytoprotection against Acrolein-Induced Carbonyl Stress

“…Low hydralazine concentrations also block the toxicity of acrolein in diverse cell types including hepatocytes, neuronal cells, and lung epithelial cells (LiuSnyder et al, 2006;Burcham et al, 2007;Truong et al, 2009). Protective efficacy for hydralazine has also been shown in several animal models of carbonyl-mediated pathology, including diabetic nephropathy, allyl alcohol hepatotoxicity, spinal cord injury, atherosclerosis, and experimental autoimmune disease (Nangaku et al, 2003;Kaminskas et al, 2004b;Vindis et al, 2006;Hamann et al, 2008;Leung et al, 2011). The mechanism(s) underlying drug efficacy in these models is unclear because the direct reactivity of hydralazine with electrophilic carbonyls may be constrained within biological systems.…”