Abstract
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 Toxicol15:40–47, 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 μM (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 μM 4-HNE(Ac)3 (cytotoxicity = 33%), the mutant frequency was 719 × 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.
Footnotes
-
This research was supported in part by U.S. Public Health Service Grant R01 ES07804 (to Piotr Zimniak), awarded by the National Institute of Environmental Health Sciences, by U.S. Public Health Service Grant P01 AG20641 awarded by the National Institute of Aging (to Robert J. Shmookler Reis; John J. Thaden, Analytical Core leader), and by appointments (Nan Mei and Ling Chen) to the Postgraduate Research Program at the National Center for Toxicological Research (NCTR) administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.
-
doi:10.1124/jpet.104.080754.
-
ABBREVIATIONS: ROS, reactive oxygen species; 4-HNE, 4-hydroxynon-2-enal; 4-HNE(Ac)3, 4-HNE triacetate; MLA, mouse lymphoma assay; TFT, trifluorothymidine; NQO, 4-nitroquinoline-1-oxide; PBS, phosphate-buffered saline; DNPH, 2,4-dinitrophenylhydrazine; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; MF, mutant frequency; RTG, relative total growth; LOH, loss of heterozygosity; Tk1, thymidine kinase; PCR, polymerase chain reaction; cM, centimorgan.
- Received November 14, 2004.
- Accepted February 3, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|