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

Abstract

Hydralazine strongly suppresses the toxicity of acrolein, a reactive aldehyde that contributes to numerous health disorders. At least two mechanisms may underlie the cytoprotection, both of which involve the nucleophilic hydrazine possessed by hydralazine. Under the simplest scenario, hydralazine directly scavenges free acrolein, decreasing intracellular acrolein availability and thereby suppressing macromolecular adduction. In a second "adduct-trapping" mechanism, the drug forms hydrazones with acrolein-derived Michael adducts in cell proteins, preventing secondary reactions of adducted proteins that may trigger cell death. To identify the most important mechanism, we explored these two pathways in mouse hepatocytes poisoned with the acrolein precursor allyl alcohol. Intense concentration-dependent adduct-trapping in cell proteins accompanied the suppression of toxicity by hydralazine. However, protective concentrations of hydralazine did not alter extracellular free acrolein levels, cellular glutathione loss, or protein carbonylation, suggesting that the cytoprotection is not due to minimization of intracellular acrolein availability. To explore ways whereby adduct-trapping might confer cytoprotection, the effect of hydralazine on acrolein-induced protein crosslinking was examined. Using bovine pancreas ribonuclease A as a model protein, acrolein caused rapid time-and concentration-dependent cross-linking, with dimerized protein detectable within 45 min of commencing protein modification. Lysine adduction in monomeric protein preceded the appearance of oligomers, whereas reductive methylation of protein amine groups abolished both adduction and oligomerization. Hydralazine inhibited cross-linking if added 30 min after commencing acrolein exposure but was ineffective if added after a 90-min delay. Adduct-trapping closely accompanied the inhibition of cross-linking by hydralazine. These findings suggest that cross-link blocking may contribute to hydralazine cytoprotection.

Chitosan produces potent neuroprotection and physiological recovery following traumatic spinal cord injury

“…Thus, the production of ROS or so-called free radicals (such as superoxide (O 2 -), hydroxyl ions (OH), and hydrogen peroxide (H 2 O 2 ), is a direct result leading to skyrocketing, secondary oxidative stress. Understandably, such endogenous and deleterious substances continue to result in progressive cell destruction via LPO and its derivatives, toxic intracellular aldehydes (Burcham et al, 2004;Burcham and Pyke, 2006;Shi et al, 2002). Since these toxins, like acrolein, can pass through intact healthy membranes, their intracellular concentration increases in the microenvironment permitting the poisoning of nearby undamaged cells (called 'bystander damage').…”

“…Thus, as with reactive oxygen species, chitosan does not show any ability to scavenge acrolein, and even the application of highly concentrated chitosan does not repair injury associated with acrolein toxicity (Fig.6). On the contrary immediate treatment with hydralazine, a well-known acrolein scavenger, permitted recovery from mitochondrial injury and abnormal oxidative metabolism (Burcham and Pyke, 2006;LiuSnyder et al, 2006). In summary, chitosan enabled the reduction of ROS and LPO production by the restoration of disrupted plasma membrane.…”

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

“…Strong adduct trapping also accompanied hydralazine protection against allyl alcohol hepatotoxicity in mice (Kaminskas et al, 2004b). How adduct trapping might confer protection was unclear, but one possibility was that hydralazine inactivated carbonyl-retaining Michael adducts before their participation in cross-linking with neighboring macromolecules (Burcham and Pyke, 2006). Contrary to this expectation however, hydralazine failed to block acrolein-induced protein crosslinking in cultured lung cells (Burcham et al, 2007).…”

“…Although hydralazine displays excellent reactivity toward "free" unsaturated aldehydes in model in vitro systems, it is unclear whether these properties apply within complex intracellular environments (Burcham and Pyke, 2006). To address this issue in the lung cell model used in the present study, we determined whether hydralazine suppressed acrolein-induced adduction of IFs, which are known targets for acrolein in lung cells (Burcham et al, 2010).…”

“…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. For example, in a hepatocyte model in which acrolein was generated enzymatically from allyl alcohol, hydralazine prevented cell death without lowering either extracellular "free acrolein" levels or the formation of protein adducts (Burcham et al, 2006). Others have also observed that hydralazine fails to block protein carbonylation in cultured cells, concluding instead that antioxidant actions mediate the cytoprotection (Mehta et al, 2009;Zheng and Bizzozero, 2010).…”