Incubation of superoxide dismutase with malondialdehyde and 4-hydroxy-2-nonenal forms new active isoforms and adducts. An evaluation of xenobiotics in fish

The effects in fish (Sparus aurata) of dieldrin, previously reported to be an inducer of peroxisomal enzymes (Pedrajas et al., Comp. Biochem. Physiol. 115C (1996) 125-131), were compared with those of clofibrate. Although die ldrin provoked the more severe peroxisomal: changes, both compounds induced oxidative stress as detected by the increased levels of microsomal thiobar bituric acid reactive substances; however the malondialdehyde (MDA) content , determined after HPLC separation of the MDA-TBA complex, was not signific antly altered. These results suggest that, besides MDA, other aldehydes wer e formed in xenobiotic-injected fish, leading us to assess the oxidative ef fects of such xenobiotics by following changes in superoxide dismutase (SOD ) pattern. New active SOD isoforms were detected by isoelectrofocusing in t he light mitochondrial (LMF) and cytosolic (CF) fractions. Most of the new SOD bands could be reproduced in vitro by incubation of fish liver cell-fre e extracts with MDA. To clarify the effects of aldehydes, Cu,Zn- and Mn-SOD isoforms were. purified and amino acid analysis was carried out. The new b ands found in LMF and CF fractions were reproduced in vitro after incubatio n of pure SODs with MDA and 4-hydroxy-2-nonenal (HNE), the new SOD bands fo rmed being coincident with the loss of Lys or His residues. Lysine residues were preferentially derivatized after treatment of Cu,Zn-SOD with MDA, but in Mn-SOD the lysine residues were modified only after treatment with MDA, while the histidine residues were modified only by HNE. No change of SOD a ctivity was detected after MDA or HNE exposure, although at the higher alde hyde concentrations used protein aggregates were formed. Therefore, the app earance of new active SOD bands, after isoelectrofocusing separation, can b e proposed as a biomarker of oxidative stress. (C) 1998 Elsevier Science Ir eland Ltd. All rights reserved.