4-Hydroxynonenal and Other Aldehydes Produced in the Liver In Vivo After Bromobenzene Intoxication

Abstract

4-Hydroxynonenal (4-HNE) has been identified as one of the most reactive products in a series of toxic aldehydes originating from lipid peroxidation of cellular membranes. The possibility that this aldehyde plays a role as one of the mediators of the cellular injury induced by pro-oxidants is currently investigated. Mice intoxicated with bromobenzene showed levels of lipid peroxidation in the liver that exceed those induced by hepatotoxic haloalkanes CCl4 and BrCCl3. Hence, we have searched for the presence of 4-HNE and other lipid peroxidation products in the liver of bromobenzene-poisoned mice. We looked for 4-HNE in liver extracts as either free aldehyde or its 2,4-dinitrophenylhydrazone derivative. Using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) we obtained well resolved peaks, corresponding to the standard aldehyde or its 2,4-dinitrophenylhydrazone derivative, respectively. 2,4-dinitrophenylhydrazone derivatization was also used to determine the total carbonyl content in the liver of the intoxicated animals. Three fractions of hydrazones, according to their different polarity (“polar”; “non-polar carbonyls, fraction I”; and “non-polar carbonyls, fraction II”), were obtained using TLC. The UV-visible spectra were recorded for quantitative evaluation. Further fractionation of “non-polar carbonyls, fraction II” provided a fraction containing several alkanals and alk-2-enals, which were analyzed and identified by HPLC. Furthermore, protein bound carbonyls were determined in the liver of the intoxicated animals.