The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) is cytotoxic and genotoxic at superphysiological concentrations. To characterize the mechanism of action of 4-HNE, we assessed genotoxic damage by 4-HNE and by 4-HNE triacetate [4-HNE(Ac)(3)] using the mouse lymphoma assay that measures the mutant frequency in the Tk gene. As a strong electrophile, 4-HNE reacts readily with nucleophilic centers on cellular components. When added extracellularly, it may react preferentially with proteins in culture medium or on the cell surface and not reach deeper cellular targets such as nuclear DNA. Therefore, 4-HNE(Ac)(3), a protected form of 4-HNE that is metabolically converted to 4-HNE in cells (Neely MD, Amarnath V, Weitlauf C, and Montine TJ, Chem Res Toxicol 15:40-47, 2002), was assayed in addition to 4-HNE. When added in serum-containing medium, 4-HNE was not mutagenic in the mouse lymphoma assay up to 38 muM (cytotoxicity = 13%). In contrast, exposure to 4-HNE(Ac)(3), which mimics intracellular formation of 4-HNE, resulted in dose-dependent induction of mutations. At 17 muM 4-HNE(Ac)(3) (cytotoxicity = 33%), the mutant frequency was 719 x 10(-6) (>7-fold higher than the spontaneous mutant frequency). Loss of heterozygosity analysis in the Tk mutants revealed that the majority of mutations induced by 4-HNE(Ac)(3) resulted from clastogenic events affecting a large segment of the chromosome. The results indicate that, in the presence of serum that approximates physiological conditions, 4-HNE generated intracellularly but not extracellularly is a strong mutagen via a clastogenic action at concentrations that may occur during oxidative stress.