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TOXICOLOGY
Department of Pharmacology and Toxicology (S.P.S., E.M., V.S., P.Z.) and Department of Geriatrics (J.J.T.), University of Arkansas for Medical Sciences, and Central Arkansas Veteran's Healthcare System (S.P.S., J.J.T., P.Z.), Little Rock, Arkansas; Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas (T.C., L.C., N.M., M.M.M.); and College of Life Science and Technology, Shanghai Jiao Tong University, Shanghai, China (L.C.)
Received November 14, 2004; accepted February 3, 2005.
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Abstract |
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) can attack all cellular constituents. It is often tacitly assumed that functionally relevant effects of ROS are due to damage to proteins and nucleic acids because such damage results in immediate functional consequences or mutations. However, ROS also target lipids. The hydroxyl radical can initiate a chain reaction that produces multiple lipid hydroperoxide molecules from a single initial event (Gutteridge and Halliwell, 1990). Since lipid hydroperoxides are oxidants capable of damaging cellular constituents, the chain reaction is in effect amplifying the initial oxidative insult. Peroxidation of polyunsaturated fatty acids gives rise, in a reaction that has been partially defined (Schneider et al., 2001, 2004; Sun and Salomon, 2004), to highly electrophilic 4-hydroxy-alkenals, of which 4-hydroxynon-2-enal (4-HNE; Fig. 1A) is the major example. 4-HNE is capable of modifying and/or cross-linking proteins (Cohn et al., 1996; Waeg et al., 1996; Morikawa et al., 1997; Sayre et al., 1997), thus altering or impairing protein function. The formation of adducts on proteins has wide-ranging and profound functional consequences not only in terms of damage to cellular macromolecules, but also in terms of regulatory effects (reviewed in Dianzani, 2003; Forman et al., 2003). In particular, early phases of stress signaling may be mediated by 4-HNE (Cheng et al., 2001). Because of the amplification inherent in the lipid peroxidation chain reaction, cellular damage and modifications linked to 4-HNE may be quantitatively more important than a direct attack by ROS.
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It has been recognized since 1993 (Esterbauer, 1993) that 4-HNE reacts with DNA, albeit less readily than with proteins, rendering the compound not only cytotoxic but also genotoxic. In endothelial cells and in primary rat hepatocytes, 4-HNE caused an increase in the frequency of sister chromatid exchange, micronuclei formation, and chromosomal aberrations at concentrations lower than those necessary for cytotoxicity (Eckl, 2003). All four bases are targets for 4-HNE adduct formation, and exposure of single-stranded phage DNA to 4-HNE led to an inhibition of DNA replication and an increased level of recombination (Kowalczyk et al., 2004).
The high chemical reactivity of 4-HNE, particularly toward nucleophiles (Schaur, 2003), poses a problem in studying the biological properties of this compound. Extracellularly added 4-HNE may react preferentially with target protein or lipid molecules at the cell surface or a zone of cytoplasm immediately underneath the plasma membrane. In such cases, the concentration of 4-HNE reaching nuclear or mitochondrial DNA would be unknown and variable, but would probably constitute only a small fraction of the 4-HNE concentration in the medium. This problem has been elegantly albeit partially solved by Neely et al. (2002) through the synthesis of 4-HNE triacetate [4-HNE(Ac)3; Fig. 1B], a diffusible and biologically inert precursor from which 4-HNE is enzymatically liberated by intracellular hydrolases. Although the protective acetyl groups are readily removed from the aldehyde function of 4-HNE, the hydroxyl group in position 4 remains largely in the form of an acetyl ester (Fig. 1C) (Neely et al., 2002). This presents a limitation to the use of 4-HNE(Ac)3 as a 4-HNE precursor since the 4-acetoxy group has weaker electron-withdrawing properties than the 4-hydroxyl group, rendering the acetylated 4-HNE a weaker electrophile than 4-HNE itself. Nevertheless, deprotection of the carbonyl group restores the conjugated double bond system of 4-HNE that is responsible for its Michael acceptor character and its reactivity toward nucleophilic biological targets.
In the present study, we compared the mutagenic effects of 4-HNE and 4-HNE(Ac)3 in the mouse lymphoma assay (MLA) and conducted an analysis of the types of induced mutations. We selected the MLA because of its ability to detect a broad spectrum of genetic damage including both point and chromosomal mutations (Hozier et al., 1981; Moore et al., 1985a,b, 2002, 2003; Applegate et al., 1990; Chen et al., 2002a,b).
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Materials and Methods |
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) and Chandra and Srivastava (1997), and 4-HNE(Ac)3 was prepared as described by Neely et al. (2002). The identity of both compounds was confirmed by proton NMR (data not shown). Trifluorothymidine (TFT), Tris, MgCl2, 4-nitroquinoline-1-oxide (NQO), Triton X-100 and Tween 20 were obtained from Sigma-Aldrich (St. Louis, MO). Fischer's Medium for Leukemic Cells of Mice, horse serum, Pluronic F68 [triblock copolymer: (ethylene oxide)75-(propylene oxide)30-(ethylene oxide)75, Ahmed et al., 2001], penicillin-streptomycin, sodium bicarbonate, sodium pyruvate, phosphate-buffered saline (PBS, 2.67 mM KCl, 1.47 mM KH2PO4, 137.93 mM NaCl, and 8.06 mM Na2HPO4), and proteinase K were purchased from Invitrogen (Carlsbad, CA). 2,4-Dinitrophenylhydrazine (DNPH) was obtained from Sigma-Aldrich, recrystallized from acetonitrile, and stored under nitrogen gas at –20°C.
Cells and Chemical Treatment. The Tk+/–-3.7.2 heterozygote of the L5178Y mouse lymphoma cell line was utilized for the mutation assay. Growth conditions, treatment, and expression were performed according to the procedures described by Chen and Moore (2004). Cells were maintained in logarithmic growth in culture using Fischer's Medium for Leukemic Cells of Mice supplemented with 10% horse serum, 100 µg/ml sodium pyruvate, 0.05% (v/v) Pluronic F68, 100 U/ml penicillin, and 100 µg/ml streptomycin. In preparation for chemical treatment, cells were centrifuged, and 6 x 106 cells were suspended in cell culture flasks in 10 ml of medium including 5% horse serum and the other supplements listed above. The test compounds 4-HNE and 4-HNE(Ac)3 were dissolved in DMSO and were added to the suspended cells at concentrations ranging from 12.8 to 38.4 µM (2.0 to 6.0 µg/ml) for 4-HNE and from 1.7 to 23.3 µM (0.5 to 7.0 µg/ml) for 4-HNE(Ac)3. NQO (the positive control) also was dissolved in DMSO and used at a final concentration of 0.53 µM (0.1 µg/ml). In all cases, including negative controls, the final concentration of DMSO in the medium was 1%. The cell culture flasks were placed in a humidified incubator gassed with 5% CO2 in air at 37°C for 4 h. At the end of the treatment period, the cells were pelleted by centrifugation, washed twice, and resuspended in fresh medium. The cells then were placed in a humidified incubator at 37°C in the presence of 5% CO2 and maintained in log-phase growth for a 2-day expression period.
Identification and Quantitation of Products of 4-HNE(Ac)3 Hydrolysis by Mouse Lymphoma Cells. L5178/Tk+/– mouse lymphoma cells (6 x 106 cells per flask in 10 ml of medium) were treated with 16.6 µM 4-HNE(Ac)3 as described above. After different incubation times (0 to 4 h), cells were pelleted, washed with PBS, and sonicated in 0.5 ml of PBS. The resulting cell lysate was treated with 0.5 ml of 1.8 mM DNPH in 1 N HCl for 2 h at room temperature followed by 1 h at 4°C according to Esterbauer and Cheeseman (1990). The reaction mixture was extracted three times with 0.5 ml of dichloromethane. Combined organic phases were evaporated to dryness in a Speedvac. Residual solids were dissolved in 60 µl of acetonitrile. Fifty microliters of this solution were injected on a Supelcosil LC-318 C18 reverse phase column (25 cm x 4.6 mm, 5-µm particle size, Supelco; Sigma-Aldrich) for HPLC analysis at a flow rate of 1 ml/min of solvent A (5% acetonitrile in water + 0.1% trifluoroacetic acid) for 2 min, followed by a linear gradient from 0 to 100% solvent B (acetonitrile + 0.1% trifluoroacetic acid) between 2 and 30 min. Peaks eluting at 23.7 and 26.4 min were quantified on the basis of peak area assuming an extinction coefficient 
340 = 25 mM–1 · cm–1 for 2,4-dinitrophenylhydrazones (Esterbauer and Cheeseman, 1990). For identification, collected peak fractions (yellow in color) were infused at a rate of 10 µl/min into the unheated Turboionspray source of an API3000 triple-quadrupole mass spectrometer (MDS/Sciex; Applied Biosystems, Foster City, CA) operating with an ion-source potential of –3700 V and with nitrogen nebulizer- and curtain-gas flows of 0.4 and 1 l/min, respectively. For tandem mass spectrometry, the collision gas also was nitrogen at an instrument setting of 6. Declustering, focusing, and entrance potentials were set to –50, –200, and –10 V, respectively, for mass and tandem mass spectrometry of the 23.7-min HPLC fraction and were set to –25, –85, and –5.7 V for analysis of the 26.4-min fraction. For in vitro hydrolysis of 4-HNE(Ac)3, the compound was dissolved in a small amount of DMSO, and the solution was diluted with PBS to yield final concentrations of 23 µM 4-HNE(Ac)3 and 5% (v/v) DMSO. Aspergillus oryzae lipase (Fluka; Sigma-Aldrich) was added to 4 mg/ml (192 units/ml), and the reaction mixture was incubated for 30 min at room temperature.
Tk Microtiter Mutation Assay. Mutant selection was performed as described by Chen and Moore (2004). Briefly, following the 2-day expression, the cells were counted and the densities adjusted using fresh medium. For mutant enumeration, TFT (3 µg/ml) was added to the cell culture and cells were seeded into four 96-well flat-bottom microtiter plates using 200 µl per well and a density of 2000 cells/well. For the determination of plating efficiency, approximately 1.6 cells were aliquoted at 200 µl per well into two 96-well flat-bottom microtiter plates. All plates were incubated at 37°C in a humidified incubator with 5% CO2 in air. After 11 days of incubation, colonies were counted and categorized as small or large. Small colonies were defined as those smaller than 25% of the well. Mutant frequencies (MF) were calculated using the Poisson distribution. Cytotoxicity was measured using relative total growth (RTG) which includes a measure of growth during treatment, expression, and cloning (Chen and Moore, 2004).
Detection of LOH at the Thymidine Kinase (Tk1) Locus and the Microsatellite D11Mit42, D11Mit29, and D11Mit74 Loci. TFT-resistant clones were directly taken from the microtiter plates. Only mutants obtained following treatment with 16.6 µM 4-HNE(Ac)3 were isolated and further analyzed. The mutants from the 4-HNE exposure were not analyzed for LOH because 4-HNE was not mutagenic under the conditions used (see Results). Cells were washed once with PBS by centrifugation, and cell pellets were quickly frozen and stored at –80°C. Genomic DNA was extracted by digesting the cells in lysis buffer [10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1% (v/v) Triton X-100, and 1% (v/v) Tween 20] with 200 µg/ml proteinase K at 60°C for 90 min, followed by inactivation of proteinase K at 95°C for 10 min. The procedure for the PCR analysis of LOH at the Tk locus was previously described (Chen et al., 2002a). For PCR analysis of LOH at other loci, the amplification reactions were carried out in a total volume of 30 µl by using 2x PCR master mix (Promega, Madison, WI) and a pair of primers for a specific locus obtained from The Jackson Laboratory (Bar Harbor, ME; http://www.informatics.jax.org). Primer sequences are listed in Table 1. The thermal cycling conditions were as follows: initial incubation at 94°C for 3 min, 40 cycles of 94°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 30 s, and a final extension at 72°C for 7 min. The amplification products were analyzed by gel electrophoresis and scored for the presence of one band (indicating LOH) or two bands (retention of heterozygosity at the given locus).
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Data Analysis. The data evaluation criteria developed by the MLA Expert Workgroup of the International Workgroup for Genotoxicity Tests (IWGT) were used for data analysis (Moore et al., 2003). For the microwell version of the MLA, a chemical is judged to be positive if there is a dose-related increase in MF and if one or more cultures show an induced mutant frequency that exceeds the global evaluation factor of 126 x 10–6. For instance, if the background mutant frequency is 81 x 10–6 (an average of the duplicate negative control cultures) then at least one of the treated cultures must have a total mutant frequency of 81 x 10–6 + 126 x 10–6 = 207 x 10–6 to be determined to be positive. If that total mutant frequency is not attained, the response is negative.
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Results |
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) has shown that 4-HNE(Ac)3 can be converted to 4-HNE (Fig. 1A) and 4-HNE monoacetate (Fig. 1C) by neuroblastoma cells. We confirmed the ability of mouse lymphoma cells used in the present work to hydrolyze 4-HNE(Ac)3. The cells were treated with 4-HNE(Ac)3, lysed, and the lysate was derivatized with DNPH and analyzed by HPLC as described under Materials and Methods. Peaks eluting at 23.7 and 26.4 min were identified as the 2,4-dinitrophenylhydrazones of 4-HNE and 4-HNE monoacetate, respectively, by mass spectrometry. The 23.7-min mass spectrum exhibited its second strongest signal at m/z 335, nominally correct as the [M-H]– molecular anion of the 2,4-dinitrophenylhydrazone of 4-HNE. In tandem mass spectrometry (collision energy = –23 V), the pattern of m/z 335 fragments was highly consistent with that produced by an authentic sample of the 2,4-dinitrophenylhydrazone of 4-HNE. For the 26.4-min fraction, the dominant spectral signal was m/z 377, as predicted for 2,4-dinitrophenylhydrazone of the monoacetate of 4-HNE (mol. wt. 378). When exposed to a collision energy of –28 V, the m/z 377 ion generated two products attributable to deacetylation, [M-CH3COOH]– (m/z 317) and CH3COO– (m/z 59), and minor products at m/z 181, 163, 109, and 152. In vitro hydrolysis of 4-HNE(Ac)3 with lipase followed by HPLC analysis of DNPH derivatives also yielded peaks eluting at 23.7 and 26.4 min which gave mass spectra consistent with those of samples derived from mouse lymphoma cells. This result further confirms the identity of 4-HNE(Ac)3 hydrolysis products.
In agreement with previous data (Neely et al., 2002), the monoacetyl ester of 4-HNE formed more rapidly than 4-HNE itself (Fig. 2). Upon exposure of 6 x 106 cells to 170 nmol of 4-HNE(Ac)3 for 1 h, approximately 1 nmol of hydrolysis products or 0.5% of the available 4-HNE(Ac)3 was associated with cells (Fig. 2). This is in qualitative agreement with the previously reported 1.7% at a significantly higher concentration of 4-HNE(Ac)3 (250 µM, Neely et al., 2002, versus 17 µM in the present study). After 4 h of incubation, approximately 3 nmol of 4-HNE monoacetate and 0.4 nmol of 4-HNE were associated with the cells (Fig. 2). Formally, this results in a calculated intracellular concentration of approximately 5 mM for 4-HNE(Ac)3 hydrolysis products, given a mean diameter of 6 µm for mouse lymphoma cells (measured microscopically) and thus an approximate volume of 1.1 x 10–7 µl/cell or 0.66 µl of total internal volume in 6 x 106 cells.
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Mutagenicity of 4-HNE was tested across a concentration range of 12.8 to 38.4 µM (Table 2; Fig. 3,A and B). The highest concentration resulted in an RTG of 13%. Although the MF at this concentration was slightly higher than the negative control, this small increase was insufficient to result in a positive determination. In contrast, 4-HNE(Ac)3 was clearly mutagenic (Table 3; Fig. 3, C and D). The MF increased in a dose-dependent manner between 1.7 and 16.6 µM 4-HNE(Ac)3 and subsequently decreased at higher 4-HNE(Ac)3 concentrations. In fact, the MF at every 4-HNE(Ac)3 concentration, except for 1.7 and 2.5 µM, was positive. At 16.6 µM, 4-HNE(Ac)3 induced a MF of 719 x 10–6 at an RTG of 33% in one experiment (see Table 3) and a MF of 761 x 10–6 at 26% RTG (see Table 2) in the 4-HNE experiment in which it was used for comparative purposes.
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DNA samples isolated from 70 large and 69 small Tk mutant colonies from cultures treated with 16.6 µM 4-HNE(Ac)3 were analyzed for LOH at the Tk1 locus using allele-specific PCR with primers listed in Table 1. This analysis revealed that all of the small colony mutants had lost the Tk allele (Table 4). This can be compared with spontaneous mutants arising in control cultures where 91% had lost the Tk allele (Harrington-Brock et al., 2003). In contrast, the percentage of LOH in 4-HNE(Ac)3-induced large colonies (93%) was distinctly different from that of the control (68%). The total percentage of LOH in mutants from 4-HNE(Ac)3-treated and control groups were 96 and 79%, respectively.
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To estimate the range of chromosomal damage by 4-HNE(Ac)3, LOH at Tk1 and three other loci along chromosome 11 was examined in DNA isolated from 20 large and 20 small mutant colonies (Table 5). Among the 40 mutants analyzed, 98% had an alteration of DNA at the Tk locus, and 85% of the mutants showed damage that reached to 6 cM in chromosome length. Twenty-three percent of the mutants had an alteration of DNA larger than half of chromosome 11 (Fig. 4). None of the mutants had an LOH involving the entire chromosome 11. The range of DNA damage induced by 4-HNE(Ac)3 in small colonies appeared more extensive than that in large mutant colonies (Table 5).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Kowalczyk et al., 2004). The capability to detect a broad spectrum of mutational events is also the primary reason for the adoption of MLA as the preferred mammalian cell gene mutation assay for regulatory decision-making (Dearfield et al., 1991; International Conference on Harmonisation, 1995; Department of Health and Human Services, 1997). L5178Y/Tk+/–-3.7.2C cells used in the MLA have only one functional copy of the Tk1 gene. Cells are exposed to potential mutagens and any resulting thymidine kinase-deficient mutants are selected using the pyrimidine analog TFT. Tk mutant clones fall into two basic size categories. Large colony mutants grow at the same rate as the Tk+/– cells and small colony mutants grow slowly. The relative frequency of the two size classes of mutant colonies is mutagen-dependent. Chemicals whose major mechanism of action is to break chromosomes (clastogens) generally induce a higher proportion of small colonies, whereas chemicals that cause point mutations induce large colony mutants (Moore et al., 1985a,b). Tk mutants can be further characterized using a combination of molecular and cytogenetic analysis to determine the types of mutations induced by specific mutagenic agents, thus providing additional insight into their mechanisms of action (Chen et al., 2002a,b).
Although both 4-HNE and 4-HNE(Ac)3 were cytotoxic in the MLA system, at equal concentrations, 4-HNE(Ac)3 was more cytotoxic than 4-HNE. In addition, only 4-HNE(Ac)3 was significantly mutagenic. Given the relatively low chemical reactivity of 4-HNE(Ac)3 and its intracellular conversion to 4-HNE and 4-HNE monoacetate both in mouse lymphoma cells (Fig. 2) and in other cell types (Neely et al., 2002), it is unlikely that the dissimilar cytotoxicity of the two compounds is due to an intrinsically different mode of action. Rather, it appears probable that the ultimate toxicant is 4-HNE (and/or 4-HNE monoacetate) and that the difference in potency between externally added 4-HNE and 4-HNE(Ac)3 is due to the concentration and site of action of this ultimate toxicant. 4-HNE is fairly reactive and preferentially partitions into the lipid (membrane) phase (Schaur, 2003). Thus, externally added 4-HNE may react predominantly with nucleophilic sites at the plasma membrane of cells. Any remaining 4-HNE that diffuses across the plasma membrane would face the cytoplasm which can be viewed as an extremely concentrated protein solution (approximately 200 mg/ml or 4 mM assuming a molecular weight of 50 kDa for an average protein; the concentration of nucleophilic sites is still higher because multiple such sites are present on most proteins). The micromolar concentrations of added 4-HNE are approximately three orders of magnitude lower than the concentration of potentially reactive nucleophilic sites in the cytoplasm. A relatively thin layer of cytoplasm may thus effectively shield deeper intracellular targets such as the nucleus from micromolar concentrations of exogenous 4-HNE. In addition, most cells contain glutathione transferases (mGSTA4-4 in the mouse) capable of conjugating 4-HNE to glutathione with high catalytic efficiency (Zimniak et al., 1994). This, together with a millimolar concentration of glutathione in the cytoplasm, creates another effective barrier against the penetration of external 4-HNE into deeper structures of cells. Indeed, it has been previously reported (Neely et al., 2002) that 4-HNE protein adducts are present largely in outer layers of the cytoplasm in cells exposed to 4-HNE, whereas in cells treated with 4-HNE(Ac)3, deeper structures are also affected. In contrast to 4-HNE, the non-reactive 4-HNE(Ac)3 readily diffuses across the plasma membrane and can become distributed throughout the cell because it is hydrolyzed by intracellular enzymes at a rate much slower than the rate of diffusion (Neely et al., 2002). Therefore, the intracellular and especially nuclear level of 
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-unsaturated carbonyl products is likely to be higher upon exposure of cells to 4-HNE(Ac)3 than upon exposure to an equal concentration of 4-HNE. In fact, from the amount of cell-associated products of 4-HNE(Ac)3 hydrolysis, it can be calculated that the intracellular concentration of these products could formally become millimolar (see Results) even though the extracellular concentration was 17 µM. The apparent accumulation of 4-HNE(Ac)3 hydrolysis products in cells is consistent with diffusion of 4-HNE(Ac)3 across the plasma membrane followed by its hydrolysis and thus entrapment and subsequent reaction with nucleophilic targets. This accumulation could account for the greater cytotoxicity of 4-HNE(Ac)3 compared with 4-HNE. The ability to generate the ultimate toxicant in close proximity to nuclear DNA is consistent with the mutagenic properties of 4-HNE(Ac)3.
An additional factor differentiating the potencies of 4-HNE and 4-HNE(Ac)3 is the fact that test compounds in MLA are added to cells in serum-containing medium (see Materials and Methods). Although the exposure time of 4-HNE to such medium prior to its addition into the cell cultures was limited to less than 3 min in the present study, a certain loss of 4-HNE but not 4-HNE(Ac)3 can be expected due to reaction with serum proteins (Barrera et al., 1991). More important, 4-HNE will continue to react with medium components during the treatment, thus limiting the effective exposure time of cells. Although this difficulty can be circumvented by the use of serum-free medium and/or by repeated additions of small amounts of 4-HNE (Barrera et al., 1991), serum deprivation may affect the cellular response to 4-HNE. This problem was avoided by using 4-HNE(Ac)3 in the present study. Thus, the lack of a consistent mutagenic effect of 4-HNE in our present work does not contradict previous literature, but may reflect a choice of assay conditions which are closer to a normal physiological situation.
In the MLA, compounds which induce point mutations tend to result in a high proportion of large mutant colonies with little LOH of the Tk allele, whereas clastogens result in a high proportion of small colony mutants and a high proportion of LOH at the Tk locus in the large-colony mutants (Harrington-Brock et al., 2003). Depending on the severity of DNA damage, LOH will also occur at other loci along chromosome 11, at a progressively decreasing frequency as the distance from the Tk allele increases. (Obviously, DNA damage occurs equally elsewhere on chromosome 11 and on other chromosomes, but only mutations that affect the Tk locus are detectable in the MLA.) The observation that LOH occurred in 93% of large colonies from 4-HNE(Ac)3-treated cultures, versus 68% in spontaneous mutants and that small colonies formed more frequently after 4-HNE(Ac)3 treatment than in control cells, indicate that 4-HNE generated intracellularly from 4-HNE(Ac)3 is a potent clastogenic mutagen. By extension, 4-HNE that forms endogenously as a consequence of lipid peroxidation in resting but especially in stressed cells should have the same effect.
In summary, we demonstrated that 4-HNE(Ac)3, which mimics intracellular generation of ,-unsaturated aldehydes including 4-HNE, is a potent mutagen in mouse lymphoma cells at concentrations at which externally added 4-HNE is not significantly mutagenic. In addition to highlighting the experimental limitations inherent to treating cells with reactive compounds such as 4-HNE, this result indicates that 4-HNE generated by lipid peroxidation within a cell may have different biological properties than 4-HNE formed externally. For example, 4-HNE formed at the site of an inflammation and diffusing into neighboring cells may not produce the same genotoxicity as 4-HNE generated intracellularly. Under our experimental conditions, both internal and external 4-HNE was cytotoxic, but only internal 4-HNE was mutagenic. Unexpectedly, a major type of mutations induced by ,-unsaturated carbonyl compounds generated intracellularly from 4-HNE triacetate were large-scale DNA changes encompassing approximately half of the length of a chromosome. Elucidation of the mechanism of the clastogenic action of 4-HNE will require further studies.
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Acknowledgements |
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Footnotes |
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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.
Address correspondence to: Dr. Piotr Zimniak, Department of Pharmacology and Toxicology, #638, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205. E-mail zimniakpiotr{at}uams.edu
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References |
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|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Ahmed F, Alexandridis P, and Neelamegham S (2001) Synthesis and application of fluorescein-labeled Pluronic block copolymers to the study of polymer-surface interactions. Langmuir 17: 537–546.[CrossRef]
Applegate ML, Moore MM, Broder CB, Burrell A, Juhn G, Kasweck KL, Lin PF, Wadhams A, and Hozier JC (1990) Molecular dissection of mutations at the heterozygous thymidine kinase locus in mouse lymphoma cells. Proc Natl Acad Sci USA 87: 51–55.
Barrera G, Di Mauro C, Muraca R, Ferrero D, Cavalli G, Fazio VM, Paradisi L, and Dianzani MU (1991) Induction of differentiation in human HL-60 cells by 4-hydroxynonenal, a product of lipid peroxidation. Exp Cell Res 197: 148–152.[CrossRef][Medline]
Chandra A and Srivastava SK (1997) A synthesis of 4-hydroxy-2-trans-nonenal and 4-(3H) 4-hydroxy-2-trans-nonenal. Lipids 32: 779–782.[CrossRef][Medline]
Chen T, Harrington-Brock K, and Moore MM (2002a) Mutant frequencies and loss of heterozygosity induced by N-ethyl-N-nitrosourea in the thymidine kinase gene of L5178Y/Tk+/–-3.7.2C mouse lymphoma cells. Mutagenesis 17: 105–109.
Chen T, Harrington-Brock K, and Moore MM (2002b) Mutant frequency and mutational spectra in the Tk and Hprt genes of N-ethyl-N-nitrosourea-treated mouse lymphoma cells. Environ Mol Mutagen 39: 296–305.[CrossRef][Medline]
Chen T and Moore MM (2004) Screening for chemical mutagens using the mouse lymphoma assay, in Optimization in Drug Discovery: In-vitro Methods (Caldwell GW ed) pp 337–352, Humana Press, Inc., Totowa, NJ.
Cheng J-Z, Sharma R, Yang Y, Singhal SS, Sharma A, Saini MK, Singh SV, Zimniak P, Awasthi S, and Awasthi YC (2001) Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress. J Biol Chem 276: 41213–41223.
Cohn JA, Tsai L, Friguet B, and Szweda LI (1996) Chemical characterization of a protein-4-hydroxy-2-nonenal cross-link: immunochemical detection in mitochondria exposed to oxidative stress. Arch Biochem Biophys 328: 158–164.[CrossRef][Medline]
Crow JP (2000) Peroxynitrite scavenging by metalloporphyrins and thiolates. Free Radic Biol Med 28: 1487–1494.[CrossRef][Medline]
Dearfield KL, Auletta AE, Cimino MC, and Moore MM (1991) Considerations in the U.S. Environmental Protection Agency's testing approach for mutagenicity. Mutat Res 258: 259–283.[Medline]
Department of Health and Human Services (1997) Genotoxicity: a standard battery for genotoxicity testing of pharmaceuticals. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use; July 16, 1997. Food and Drug Administration, Rockville, MD.
Dianzani MU (2003) 4-Hydroxynonenal from pathology to physiology. Mol Aspects Med 24: 263–272.[CrossRef][Medline]
Eckl PM (2003) Genotoxicity of HNE. Mol Aspects Med 24: 161–165.[CrossRef][Medline]
Esterbauer H (1993) Cytotoxicity and genotoxicity of lipid-oxidation products. Am J Clin Nutr 57: 779S–786S.
Esterbauer H and Cheeseman KH (1990) Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol 186: 407–421.[Medline]
Forman HJ, Dickinson DA, and Iles KE (2003) HNE—signaling pathways leading to its elimination. Mol Aspects Med 24: 189–194.[CrossRef][Medline]
Gree R, Tourbah H, and Carrie R (1986) Fumaraldehyde monodimethyl acetal: an easily accessible and versatile intermediate. Tetrahedron Lett 27: 4983–4986.[CrossRef]
Gutteridge JMC and Halliwell B (1990) The measurement and mechanism of lipid peroxidation in biological systems. Trends Biochem Sci 15: 129–135.[CrossRef][Medline]
Harrington-Brock K, Collard DD, and Chen T (2003) Bromate induces loss of heterozygosity in the thymidine kinase gene of L5178Y/Tk+/–-3.7.2C mouse lymphoma cells. Mutat Res 537: 21–28.[Medline]
Hozier J, Sawyer J, Moore M, Howard B, and Clive D (1981) Cytogenetic analysis of the L5178Y/TK+/– leads to TK–/– mouse lymphoma mutagenesis assay system. Mutat Res 84: 169–181.[Medline]
International Conference on Harmonisation (1995) Topic S2A Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Harmonised Tripartite Guideline CPMP/ICH/141/95, approved September, 1995 (see website http://www.ifpma.org).
Kowalczyk P, Ciesla JM, Komisarski M, Kusmierek JT, and Tudek B (2004) Long-chain adducts of trans-4-hydroxy-2-nonenal to DNA bases cause recombination, base substitutions and frameshift mutations in M13 phage. Mutat Res 550: 33–48.[Medline]
Moore MM, Clive D, Howard BE, Batson AG, and Turner NT (1985a) In situ analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/– mouse lymphoma cells. Mutat Res 151: 147–159.[CrossRef][Medline]
Moore MM, Clive D, Hozier JC, Howard BE, Batson AG, Turner NT, and Sawyer J (1985b) Analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/– mouse lymphoma cells. Mutat Res 151: 161–174.[Medline]
Moore MM, Honma M, Clements J, Bolcsfoldi G, Cifone M, Delongchamp R, Fellows M, Gollapudi B, Jenkinson P, Kirby P, et al. (2003) Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report–Plymouth, UK 2002. Mutat Res 540: 127–140.[Medline]
Moore MM, Honma M, Clements J, Harrington-Brock K, Awogi T, Bolcsfoldi G, Cifone M, Collard D, Fellows M, Flanders K, et al. (2002) Mouse lymphoma thymidine kinase gene mutation assay: follow-up International Workshop on Genotoxicity Test Procedures, New Orleans, Louisiana, April 2000. Environ Mol Mutagen 40: 292–299.[CrossRef][Medline]
Morikawa S, Kurauchi O, Tanaka M, Yoneda M, Uchida K, Itakura A, Furugori K, Mizutani S, and Tomoda Y (1997) Increased mitochondrial damage by lipid peroxidation in trophoblast cells of preeclamtic placentas. Biochem Mol Biol Int 41: 767–775.[Medline]
Neely MD, Amarnath V, Weitlauf C, and Montine TJ (2002) Synthesis and cellular effects of an intracellularly activated analogue of 4-hydroxynonenal. Chem Res Toxicol 15: 40–47.[CrossRef][Medline]
Sayre LM, Zelasko DA, Harris PLR, Perry G, Salomon RG, and Smith MA (1997) 4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer's disease. J Neurochem 68: 2092–2097.[Medline]
Schaur RJ (2003) Basic aspects of the biochemical reactivity of 4-hydroxynonenal. Mol Aspects Med 24: 149–159.[CrossRef][Medline]
Schneider C, Porter NA, and Brash AR (2004) Autoxidative transformation of chiral 
6 hydroxy linoleic and arachidonic acids to chiral 4-hydroxy-2E-nonenal. Chem Res Toxicol 17: 937–941.[CrossRef][Medline]
Schneider C, Tallman KA, Porter NA, and Brash AR (2001) Two distinct pathways of formation of 4-hydroxynonenal: mechanisms of nonenzymatic transformation of the 9- and 13-hydroperoxides of linoleic acid to 4-hydroxyalkenals. J Biol Chem 276: 20831–20838.
Sun M and Salomon RG (2004) Oxidative fragmentation of hydroxy octadecadienoates generates biologically active 
-hydroxyalkenals. J Am Chem Soc 126: 5699–5708.[CrossRef][Medline]
Waeg G, Dimsity G, and Esterbauer H (1996) Monoclonal antibodies for detection of 4-hydroxynonenal modified proteins. Free Radic Res 25: 149–159.[Medline]
Zimniak P, Singhal SS, Srivastava SK, Awasthi S, Sharma R, Hayden JB, and Awasthi YC (1994) Estimation of genomic complexity, heterologous expression and enzymatic characterization of mouse glutathione S-transferase mGSTA4–4 (GST 5.7). J Biol Chem 269: 992–1000.
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