Acrolein is a highly toxic aldehyde involved in a number of diseases as well as drug-induced toxicities. Its pronounced toxicity reflects the readiness with which it forms adducts in proteins and DNA. As a bifunctional electrophile, initial reactions between acrolein and protein generate adducts containing an electrophilic center that can participate in secondary deleterious reactions (e.g., cross-linking). We hypothesize that inactivation of these reactive protein adducts with nucleophilic drugs may counteract acrolein toxicity. Because we previously observed that 1-hydrazinophthalazine (hydralazine) strongly diminishes the toxicity of the acrolein precursor allyl alcohol, we explored the possibility that hydralazine targets reactive acrolein adducts in proteins. We report that hydralazine abolished the immunoreactivity of an acrolein-modified model protein (bovine serum albumin), but only if the drug was added to the protein within 30 min of commencing modification by acrolein. The ability of a range of carbonyl-trapping drugs to interfere with "early" events in protein modification strongly correlated with their protective potencies against allyl alcohol toxicity in hepatocytes. In mass spectrometry studies using a model lysine-containing peptide, hydralazine rapidly formed hydrazones with Michael adducts generated by acrolein. Using an antibody raised against such ternary drug-acroleinprotein complexes in Western blotting experiments, clear adduct-trapping was evident in acrolein-preloaded hepatocytes exposed to cytoprotective concentrations of hydralazine ranging from 2 to 50 M. These novel findings begin to reveal the molecular mechanisms whereby hydralazine functions as an efficient "protein adduct-trapping" drug.
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.
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.
A crude extract from the Australian desert plant Eremophila neglecta has recently been shown to possess antibacterial activity in a survey of candidate plants that may bear novel antimicrobial compounds. Bioassay-directed fractionation of the Et(2)O extract of E. neglecta using a broth microdilution assay led to the isolation of three new serrulatane-type diterpenoids, 2,19-diacetoxy-8-hydroxyserrulat-14-ene (2), 8,19-dihydroxyserrulat-14-ene (3), and 8-hydroxyserrulat-14-en-19-oic acid (4), and a known o-naphthoquinone commonly referred to as biflorin (5). The structures of 2-5 were determined using 1D and 2D NMR, FTIR, and high-resolution mass spectrometry. Compounds 3-5 showed antimicrobial activity against Gram-positive bacteria including Staphylococcus aureus, Streptococcus pyogenes, and S. pneumoniae. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) ranged from 6.5 to 101.6 microM and 12.7 to 202.9 microM, respectively. No activity was observed for these compounds against Gram-negative bacteria.
The nucleophilic drug hydralazine strongly inhibits cell toxicity mediated by acrolein, a short chain 2-alkenal formed during lipid peroxidation. We here report the chemistry of acrolein-trapping by hydralazine, and show that together with its structural analogue dihydralazine, it also readily traps crotonaldehyde. Isolable reaction products included (1E)-acrylaldehyde phthalazin-1-ylhydrazone (E-APH), (1Z)-acrylaldehyde phthalazin-1-ylhydrazone (Z-APH), (1E,2E)-but-2-enal phthalazin-1-ylhydrazone (E-BPH) and (1Z,2E)-but-2-enal phthalazin-1-ylhydrazone (Z-BPH). Concentration-dependent formation of (1E)-acrylaldehyde phthalazin-1-ylhydrazone was observed in the culture media of cells co-exposed to hydralazine and the acrolein precursor allyl alcohol. These aldehyde-sequestering properties of hydrazinophthalazine drugs may contribute to the protection they provide against 2-alkenal-mediated toxicity.
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