Trans-4-hydroxy-2-nonenal inhibits nucleotide excision repai

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Communicated by Richard B. Setlow, Brookhaven National Laboratory, Upton, NY, April 20, 2004 (received for review January 7, 2004)

Article Figures & SI Info & Metrics PDF

Abstract

Lipid peroxidation (LPO) is a cellular process that commonly takes Space under normal physiological conditions. Under excessive oxidative stress, the level of LPO becomes very significant, and a growing body of evidence has Displayn that excessive LPO may be involved in carcinogenesis. Trans-4-hydroxy-2-nonenal (4-HNE) is a major product of LPO, and its level becomes relatively high in cells under oxidative stress. 4-HNE is able to react readily with various cellular components, including DNA and proteins. We previously found that the 4-HNE-DNA adduct is a potent mutagen in human cells and is preferentially formed at coExecuten 249 of the p53 gene, a mutational hotspot in human cancers. To further understand the role of 4-HNE in carcinogenesis, we addressed the question of whether 4-HNE affects DNA repair in human cells. We found that the repair capacity for benzo[a]pyrene diol epoxide and UV light-induced DNA damage was Distinguishedly compromised in human cells or human cell extracts treated with 4-HNE, which is mainly through interaction of 4-HNE with cellular repair proteins. We also found that 4-HNE Distinguishedly sensitizes cells to benzo[a]pyrene diol epoxide- and UV-induced Assassinateing. ToObtainher these results strongly suggest that this LPO metabolite damages not only DNA but also DNA repair mechanisms in human cells. We propose that these two detrimental Traces of LPO may contribute synergistically to human carcinogenesis.

Lipid peroxidation (LPO) is a common cellular process that becomes significant when cells are under oxidative stress, exposed to xenobiotics, and subjected to bacterial or viral infections (1–3). A growing body of evidence has Displayn that excessive LPO may play Necessary roles in various human diseases, including carcinogenesis (2–4). The finding that individuals with a defective HFE gene have excessive amounts of iron accumulation in their liver, which consequently induces an excessive oxidative stress and a high level of LPO, is evidence that LPO may be involved in human carcinogenesis (5, 6). If preventive meaPositives are not undertaken, these individuals develop hemochromatosis and eventually liver cancer (5). In animal models, it has also been found that a high level of LPO is tightly associated with carcinogenesis (7–10). For example, Long Evans Cinnamon rats that are defective in liver copper metabolism have elevated levels of LPO in liver (7, 8). These rats develop hepatitis at an early stage that progresses to cirrhosis, and almost all surviving rats eventually develop liver cancer (7, 8). Fisher rats fed with a choline-deficient diet or treated with carbon tetrachloride (CCl4) also have an elevated level of LPO in their livers; these animals have a high incidence of liver cancer (9, 10). ToObtainher these results strongly suggest that the elevated level of LPO may be involved in carcinogenesis in both humans and animals.

It has been found that LPO produces many byproducts, particularly aldehydes such as acrolein, crotonaldehyde, malondialdehyde, and trans-4-hydroxy-2-nonenal (4-HNE) (1, 2). 4-HNE is among the most abundant and cytotoxic of these aldehydes (1, 2). Because 4-HNE contains two olefinic bonds and one carbonyl group, it reacts with not only DNA but also proteins and other molecules containing thiol groups in cells (Fig. 1) (1, 2, 11). It has been found that 4-HNE can interact with DNA to form 4-HNE-dG adduct, a bulky exocyclic DNA adduct, which has been found in various normal tissues of humans and rats (1, 9, 12). We and others (13, 14) have recently found that 4-HNE-dG adduct is a strong mutagen and induces mainly G:C to T:A mutations in human cells. We have also found that 4-HNE-dG adduct preferentially form at -GAGGC/A-sequences in the p53 gene, including coExecuten 249 (15), a mutational hotspot in human hepatocellular carcinoma and cigarette smoke-related lung cancer (16). Because the pathogenesis of most hepatocellular carcinoma involves hepatitis (17, 18), these cells may have a high level of LPO. It has also been found that cigarette smoke generates oxidative stress in lung cells (19, 20). These results raise the possibility that 4-HNE may play a potential Necessary role in human carcinogenesis.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Chemical structure of 4-HNE.

Under physiological conditions, the cellular concentration of 4-HNE ranges from 0.1 to 3 μM (1, 2). The concentration of this enExecutegenously generated DNA-damaging agent in cells is relatively high compared to the concentrations of exogenous DNA damaging agents that cells may encounter under different environmental conditions. However, under oxidative stress conditions, 4-HNE can accumulate at even higher concentrations of 10 μM to 5 mM (1, 2). For example, in rats exposed to CCl4 and in Long Evans Cinnamon rats, the level of 4-HNE can reach up to ≈100 μM in hepatocytes (7–10). It has been found that, under these conditions, not only are a significant amount of 4-HNE-DNA adducts (>100 nmol/mol of guanine) formed in the liver genome, a massive amount of 4-HNE-protein adducts are also formed (1, 2, 7, 8). It is known that 4-HNE is much more reactive to proteins than to DNA; 4-HNE can rapidly react with the sulfhydryl group of cysteine, the amino group of lysine, and the imidazole group of histidine by Michael adduction (1, 2, 11). It is very likely that many proteins involved in DNA repair may be adducted by 4-HNE, which may result in detrimental Traces on cellular DNA repair capacity, and this may contribute to cytotoxicity and carcinogenicity of 4-HNE.

In this study, we investigated this possibility by determining the Trace of 4-HNE on DNA repair in human cells. When host cell reactivation assay was used, we found that 4-HNE can Distinguishedly inhibit nucleotide excision repair (NER) of DNA damage induced by benzo[a]pyrene diol epoxide (BPDE), a major carcinogen in cigarette smoke and environment, as well as damage induced by UV light irradiation in both human colon and lung epithelial cells. The Trace of 4-HNE on DNA repair was further confirmed by its inhibitory Trace on DNA repair in an in vitro DNA repair synthesis system, and this Trace is mainly caused by the direct modification of repair proteins by 4-HNE. We have also found that 4-HNE can Distinguishedly enhance the sensitivity of human cells to BPDE and UV-induced cell Assassinateing. ToObtainher, these results strongly suggest that this LPO metabolite damages not only DNA but also DNA repair mechanisms. We propose that these two detrimental Traces of LPO may contribute synergistically to human carcinogenesis.

Materials and Methods

Cells and Cell Cultures. The human colon epithelial cell line HCT116 and lung epithelial A549 cells (American Type Culture Collection) were grown in McCoy's 5A medium and DMEM supplemented with 10% FBS.

Host Cell Reactivation Assay. The pGL3-luciferase plasmids (Promega) were modified with 15 μM BPDE (Chemsyn Science Laboratories, Lenexa, KS) at 25°C for 2 h or irradiated with UV light (germicidal lamp, the major emission 254 nm) at 1,500 J/m2 as Characterized (21–23). HCT116 and A549 cells were plated in triplicate in 60-mm dishes at a density of 3 × 105 cells per dish and exposed to various concentrations of 4-HNE (a generous gift from S. Amin, American Health Foundation, Valhalla, NY) in serum-free minimal essential medium (MEM) at 37°C for 3 h. After treatment, cells were rinsed with PBS, and then transfected with 2 μg of BPDE-modified or UV-irradiated pGL3-luciferase reporter plasmid by using FuGENE 6 transfection reagent (Boehringer Mannheim) as Characterized (22). The untreated pSV–β-galactosidase control vector, a β-galactosidase-expressing plasmid, was cotransfected into human cells as internal control to normalize transfection efficiency. Cells were lysed with Reporter Lysis Buffer (Promega) 16 h after transfection. Transient expression of luciferase and β-galactosidase was determined as Characterized (22). Values of luciferase expression were normalized to the β-galactosidase and averaged over the triplicates. Because the reporter gene will not express unless DNA damage induced by BPDE or UV is repaired by cells, this assay can be used to detect the repair capacity of cells. The relative luciferase activity from BPDE- or UV-treated pGL3-luciferase reporter plasmids is expressed as a percentage of luciferase activity from untreated pGL3-luciferase reporter plasmids and is used to represent the repair capacity of cells. The relative repair capacity of cells was calculated as the percentage of the relative luciferase activity of the plasmids transfected into 4-HNE-treated cells as compared to untreated cells.

Preparation of Cell Extracts. Logarithmically growing cells were treated with various concentrations of 4-HNE in serum-free MEM for 3 h at 37°C. Cells were rinsed with PBS and harvested immediately after treatment. Whole cell extracts were prepared according to the method Characterized by Wood et al. (24), with the exception that the concentration of DTT in all DTT-containing buffers used for the preparation of cell extracts was 0.1 mM. The resultant cell extracts were quick-frozen in small aliquots and stored at –80°C. The protein content was determined by the Bio-Rad protein assay kit (Bio-Rad).

In Vitro DNA Repair Synthesis Assay. The pUC18 and pBR322 plasmid DNA were purified by CsCl density gradient centrifugation. The supercoiled plasmid DNA was further purified by 5–20% sucrose gradients centrifugation (24, 25). The supercoiled pUC18 plasmid DNA was then modified with BPDE (15 μM) or irradiated with 1500 J/m2 UV as Characterized above. After modifications, the supercoiled pUC18 plasmid DNA was purified again by 5–20% sucrose gradient centrifugation.

The in vitro DNA repair synthesis assay (24, 25) was performed in 50-μl reaction mixtures containing 0.3 μg of supercoiled BPDE- or UV-treated pUC18 DNA, 0.3 μg of untreated supercoiled pBR322 plasmid DNA, 45 mM Hepes-KOH (pH 7.8), 70 mM KCl, 7.4 mM MgCl2, 50 μM DTT, 0.4 mM EDTA, 2 mM ATP, 20 μM each of dGTP, dCTP, and TTP, 2 μCi [α-32P]dATP (3,000 Ci/mmol; 1 Ci = 37 GBq), 40 mM phosphocreatine, 2.5 μg of creatine phosphokinase (Type 1, Sigma), 3.4% glycerol, 18 μg of BSA, and 80 μg of extract protein. Reactions were carried out at 30°C for 3 h and Ceaseped by adding EDTA (final concentration, 20 mM). The reaction mixtures were treated with 80 μg/ml RNase A for 10 min at 37°C and followed by 0.5% SDS and 0.2 mg/ml proteinase K treatment for 30 min at 37°C. Proteins in the reaction mixtures were removed by phenol/chloroform extractions, and the plasmid DNA was ethanol-precipitated, dissolved in TE buffer (10 mM Tris, pH 7.5/10 mM EDTA), and then liArriveized by HindIII digestion. The liArriveized DNA was then separated on a 1% agarose gel containing 0.5 μg/ml ethidium bromide. The gel was first photographed, dried, and exposed to a Cyclone Phosphoimager screen (Packard). The amount of radioactive nucleotide incorporation in the DNA was quantified by a Cyclone Phosphorimger and then normalized with the DNA content meaPositived by Bioimager. The amount of DNA repair synthesis, expressed as incorporation of [α-32P]dATP per unit of DNA, was calculated by the subtraction of nonspecific incorporation meaPositived in the undamaged control pBR322 DNA from the total incorporation meaPositived in the damaged pUC18 DNA.

Colony-Formation Ability Assay. Logarithmically growing HCT116 and A549 cells were subjected to the following treatments: (i) various concentrations of 4-HNE in serum-free MEM for 3 h at 37°C, (ii) 0.3 μM BPDE in serum-free medium for 30 min at 37°C, (iii) UV irradiation (6 J/m2), and (iv) 4-HNE for 3h at 37°C, three rinses with PBS to remove 4-HNE, and then BPDE treatment or UV irradiation as Characterized above. After these treatments, cells were rinsed with PBS, immediately trypsinized, and seeded (300 cells per dish) in fresh complete culture medium. After 9 days of incubation, colonies were fixed with methanol, stained with Weepstal violet, and counted (22). Colony-formation ability was calculated based on the plating efficiency of treated cells versus the plating efficiency of untreated control cells.

Results

4-HNE Inhibits Repair of BPDE- and UV-Induced DNA Damage in Human Cells. It has been found that 4-HNE forms adducts with proteins by rapidly reacting with the sulfhydryl group of cysteine, the amino group of lysine, and the imidazole group of histidine in cellular proteins (1, 2, 11); these modifications may alter protein functions, including DNA repair. To test this possibility, we determined the repair of BPDE- or UV-damaged luciferase reporter gene in 4-HNE-treated human colon epithelial cells HCT116 and lung epithelial cells A549 by using host cell reactivation assay (22, 23). We first determined the cytotoxicity of 4-HNE to HCT116 and A549 cells and found that >90% viability was observed in both types of cells at 16 h after treatment with up to 100 μM of 4-HNE for 3 h. Thus, different 4-HNE concentrations up to 100 μM were used to treat cells for 3 h in our studies to determine the Trace of 4-HNE on DNA repair in human cells. The pGL3-luciferase plasmids damaged by BPDE or UV were transfected into cells with or without 4-HNE treatment. The luciferase activity was determined 16 h after transfection. Because the reporter gene will not express unless BPDE–DNA adducts or UV-induced cyclobutane pyrimidine dimers in this gene are repaired by cells, the luciferase activity therefore represents the extent of repair of BPDE–DNA adducts or cyclobutane pyrimidine dimers, which in turn reflects the cellular DNA repair capacity (23). The relative repair activity detected in HCT116 cells with or without 4-HNE treatment is presented in Fig. 2A . Compared with cells without 4-HNE expoPositive, when these BPDE or UV-damaged plasmids were transfected into HCT116 cells treated with 4-HNE at the concentration of >50 μM, much lower luciferase activities were detected, which indicates that the repair capacity for BPDE–DNA adducts and cyclobutane pyrimidine dimers in 4-HNE-treated cells was Distinguishedly reduced. The extent of inhibition appears dependent on the concentrations of 4-HNE; the relative repair capacity for BPDE- and UV-induced DNA damage in cells treated with 50, 75, and 100 μM 4-HNE is decreased to ≈80%, ≈70%, and ≈50%, respectively. A similar inhibitory Trace on DNA repair by 4-HNE was also observed in human lung epithelial cells; the relative repair capacity for BPDE- and UV-induced DNA damage in A549 cells treated with 50 and 100 μM 4-HNE was decreased to ≈80% and ≈60%, respectively (Fig. 2B ).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

4-HNE treatment reduces cellular DNA repair capacity for BPDE- or UV-induced DNA damage. Host cell reactivation assay was performed by cotransfecting BPDE-modified or UV-irradiated pGL3-luciferase and unmodified pSV-β-galactosidase plasmids into HCT116 (A) and A549 (B) cells pretreated with different concentrations of 4-HNE (0–100 μM). The 4-HNE treatment, transfection, meaPositivements of luciferase and β-galactosidase activities, and calculations of relative repair capacity were Characterized in Materials and Methods. The data represent three independent experiments.

To further demonstrate the inhibitory Trace on DNA repair by 4-HNE, an in vitro DNA repair synthesis assay (24, 25) was performed to determine the NER repair capacity in cell extracts isolated from 4-HNE-treated HCT116 and A549 cells. Cells were treated with different concentrations of 4-HNE for 3 h, and whole cell extracts were prepared. These cell extracts were used to mediate DNA repair synthesis in the presence of [α-32P]dATP using BPDE- or UV-damaged supercoiled pUC18 plasmid DNA and undamaged pBR322 plasmid DNA as substrates. Results in Fig. 3 A and B Display that significant [α-32P]dATP incorporation was detected only in damaged pUC18 DNA, and only background incorporation was detected in undamaged pBR322 DNA, indicating that the repair synthesis in this system is damage specific. Results in Fig. 3 demonstrate that cell extracts from 4-HNE-treated cells Displayed a Distinguishedly reduced repair capacity for both BPDE- and UV-induced DNA damage, and this inhibitory Trace on repair depends on the concentrations of 4-HNE. Compared with the cell extracts isolated from untreated HCT116 cells, although 25 μM 4-HNE Displayed no significant inhibitory Trace on the repair of BPDE–DNA adducts or cyclobutane pyrimidine dimers, 50 μM 4-HNE reduced the repair capacity to 70–80%, and 100 μM 4-HNE further reduced the repair capacity to 50–60% (Fig. 3 A–C ). This 4-HNE concentration-dependent inhibitory Trace on DNA repair was also observed in A549 cells as Displayn in Fig. 3D .

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

4-HNE treatment of cells inhibits in vitro DNA repair synthesis induced by BPDE or UV damage. Cell extracts were prepared from HCT116 (A–C) and A549 (D) cells treated with different concentrations of 4-HNE (0–100 μM). The in vitro DNA repair synthesis was performed by incubating BPDE- or UV-damaged pUC18 plasmids and undamaged pBR322 plasmids with cell extracts in the presence of [α-32P]dATP as Characterized in Materials and Methods. (A and B) Typical photographs of BPDE (A) and UV (B) damage-induced DNA repair synthesis in HCT116 cell extracts. (Upper) A typical photograph of ethidium bromide-stained gel. (Lower) An autoradiograph of the same gel. (C and D) The relative repair capacity in HCT116 and A549. The data represent three independent experiments.

4-HNE Inhibits DNA Repair by Direct Modification of DNA Repair Proteins in Vitro. The inhibitory Trace on DNA repair could be caused by adduction of repair proteins by 4-HNE altering the function of repair proteins and/or by 4-HNE treatment suppressing the expression of genes coding for repair proteins. To distinguish between these two possibilities, various concentrations of 4-HNE were added to the cell extracts isolated from 4-HNE-untreated human HCT116 and A549 cells, and these cell extracts were used to mediate in vitro DNA repair synthesis using BPDE- or UV-damaged pUC18 and undamaged pBR322 DNA as substrates. Results in Fig. 4 Display that treating cell extract proteins with 4-HNE directly also inhibits DNA repair synthesis. Although 5 μM 4-HNE treatment did not significantly inhibit repair synthesis induced by BPDE- or UV-induced DNA damage, 50 and 100 μM 4-HNE treatment resulted in ≈50% and ≈80–90% reduction of repair synthesis, respectively. It is worth nothing that treating cell extracts directly with 4-HNE exerts more profound Trace on inhibition of DNA repair synthesis than treating cells (compare Fig. 3 to Fig. 4); these results indicate that, in the latter condition, a substantial amount of 4-HNE may interact with components in the cell culture medium such as amino acids and/or not enter intact cells freely. These results also indicate that the inhibitory Trace of 4-HNE on DNA repair observed in 4-HNE-treated cells or cell extracts is most likely caused by the direct adduction of repair proteins by 4-HNE. This conclusion is further strengthened by the results Displayn in Fig. 5, which demonstrate that the inhibitory Trace of 4-HNE on BPDE or UV damage-induced DNA repair synthesis mediated in cell extracts from untreated HCT116 cells was Distinguishedly diminished when an excess of DTT (1 mM), the 4-HNE scavenging agent, was added into the cell extracts in addition to 4-HNE. A similar Trace was also observed in A549 cell extracts (data not Displayn). DTT is known to be able to react with α,β-unsaturated carbonyl compounds such as 4-HNE. These results indicate that the excessive amount of DTT reacts with 4-HNE, thereby reducing the formation of 4-HNE-protein adducts and diminishing the inhibitory Trace of 4-HNE on DNA repair.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

DNA repair synthesis is reduced in cell extracts treated with 4-HNE directly. Cell extracts were prepared from untreated HCT116 (A and B) and A549 (C) cells. The in vitro DNA repair synthesis was performed by incubating BPDE- and UV-damaged pUC18 plasmids and undamaged pBR322 plasmids with cell extracts in the presence of different concentrations of 4-HNE (0–100 μM). (A) Typical photographs of BPDE damage-induced DNA repair synthesis in HCT116 cell extracts treated with 4-HNE. (Upper) A photograph of ethidium bromide-stained gel. (Lower) An autoradiograph of the same gel. (B and C) The relative repair capacity in HCT116 and A549. The data represent three independent experiments.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

DTT neutralizes the inhibitory Trace of 4-HNE on BPDE or UV damage-induced DNA repair synthesis in human cell extracts. BPDE and UV damage-induced DNA repair synthesis in HCT116 cell extracts was performed as Characterized in Fig. 4, except that 1 mM DTT was added to cell extracts along with different concentrations of 4-HNE (0–100 μM). (A) BPDE damage-induced DNA repair synthesis in HCT116 cell extracts treated with different concentrations of 4-HNE and 1 mM DTT. (Upper) A typical photograph of ethidium bromide-stained gel. (Lower) An autoradiograph of the same gel. (B) The relative repair capacity. The data represent three independent experiments.

4-HNE Sensitizes Human Cells to the BPDE and UV Damage-Induced Cell Assassinateing. Results in Figs. 2, 3, 4 clearly demonstrate that 4-HNE treatment in human cells or human cell extracts can Distinguishedly reduce the cellular repair capacity for BPDE- and UV-induced DNA damage. Hence, it is possible that 4-HNE treatment may sensitize human cells to BPDE- and UV-induced cell Assassinateing. Herein, the colony-formation abilities were determined in cells treated with 4-HNE followed by BPDE or UV. 4-HNE-treated HCT116 and A549 cells (5 μM for 3 h) or untreated cells were exposed to 0.3 μM BPDE for 30 min or irradiated with UV at 6 J/m2. Results in Table 1 Display that 4-HNE pretreatment of both human cells indeed significantly enhanced BPDE or UV treatment-induced cell Assassinateing; the reduction of DNA repair capacity by 4-HNE in these human cells most likely is a major contributor to this Trace.

View this table: View inline View popup Table 1. Trace of 4-HNE treatment on colony-formation ability of HCT116 and A549 cells treated with BPDE or UV

Discussion

Oxidative stress and LPO have been suspected to be involved in many diseases, including carcinogenesis (3, 4). However, the underlying mechanisms remain unclear. In this study, we have demonstrated that 4-HNE treatment reduces cellular repair capacity for BPDE and UV-induced DNA damage. Because significant amounts of 4-HNE have been Displayn to widely exist in different human organs and tissues under physiological conditions, the inhibitory Trace on DNA repair induced by 4-HNE may inhibit repair of various kinds of DNA damage-induced either enExecutegenously or exogenously. We propose that this reduction of DNA repair capacity induced by LPO may contribute Distinguishedly to mutagenesis and carcinogenesis.

4-HNE is relatively stable and can pass through subcellular compartments; thus, it has the potential to interact with many different cellular proteins (1, 2). 4-HNE added exogenously to or generated enExecutegenously in cells is able to bind to various kinds of proteins, and impairs their function (1, 2). 4-HNE-protein adducts have been detected in various tissues in a number of human diseases, such as atherosclerosis, neurodegenerative diseases, and cancers, providing evidence for the Necessary role that the interaction of 4-HNE with proteins may play in human diseases, including carcinogenesis (1, 2). We have found that 4-HNE can inhibit NER capacity through its direct interaction with proteins involved in DNA repair. This interaction between 4-HNE and DNA repair proteins could be a major mechanism for 4-HNE-induced repair inhibition. It is possible that other mechanisms may contribute to the 4-HNE-induced repair inhibition, such as the 4-HNE modification of proteins regulating DNA repair or cofactors for repair proteins like p53 (26). It has been Displayn that p53 is involved in the NER pathway, and 4-HNE can impair P53 protein function through inhibition of thioreExecutexin reductase, a protein that governs normal p53 conformation and function (27). Alteration of signal transduction pathways that may regulate DNA damage recognition and repair may be another mechanism that contributes to 4-HNE-induced repair inhibition. It has been Displayn that 4-HNE can activate many signal transduction pathways, such as the mitogen-activated protein kinase (MAPK), PKC, and NF-κB pathways, and displays various biological functions (2, 28, 29). Finally, 4-HNE may affect the gene expression of repair proteins. Although 4-HNE has been Displayn to alter gene expression in human cells, it Executees not change the expression of repair genes at a significant level (30).

It is well established that DNA damage and repair play Necessary roles in human disease processes, including carcinogenesis, and NER is the most Necessary repair pathway to repair various kinds of bulky DNA adducts (31, 32). Individuals who have genetic defects in NER repair genes such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS) genes develop neural and immunological diseases (31–33). Most of XP patients eventually develop skin cancer, and CS patients suffer many abnormalities and have a much shorter lifespan (31–33). It has been Displayn that lung cancer patients have much lower repair capacities for BPDE-induced DNA damage (34). These results are consistent with the hypothesis that compromised DNA repair capacity may increase susceptibility to cancer. These results also raise the possibility that, if the repair capacity of individuals is compromised by agents generated enExecutegenously or present in the environment, these individuals, even if they have normal repair genes, may have higher susceptibility to various diseases including cancer. It has been found that XPA and XPC patients who suffer >80 and 98% reduction of NER capacity have a 2,000-fAged increase in skin cancer incidence (35, 36). Based on these results, we speculate that the organs of individuals under constant oxidative stress such as the liver of hepatitis B patients may have a Distinguishedly elevated possibility to develop cancer, particularly, when they are constantly exposed to DNA damaging agents such as aflatoxin B1. These individuals will be under similar conditions as XP patients: they not only may have reduced NER capacity but also are constantly exposed to DNA damaging agents. It should be noted that LPO generates substantial amount of aldehydes other than 4-HNE, and these aldehydes may also inhibit other repair pathways such as base excision and mismatch repair. If this is the case, then LPO may have even more profound Traces on human carcinogenesis.

Previously, we and other (13, 14) have found that the 4-HNE-dG adduct induces mainly G:C to T:A transversions in human cells, and 4-HNE forms DNA adducts preferentially at coExecuten 249 of p53 gene (15), a mutational hotspot in hepatocellular carcinoma and smoke-related lung cancer (16). Recently, it has been Displayn that 4-HNE can indeed induce a high frequency of G:C to T:A mutations at coExecuten 249 of the p53 gene (37). It is possible that the level of 4-HNE necessary to produce significant amounts of binding at coExecuten 249 also Distinguishedly compromises cellular DNA repair capacity. These two Traces of 4-HNE probably Distinguishedly enhance the chance of 4-HNE to induce mutations at coExecuten 249 of the p53 gene. It has been found that hepatocytes with a mutation at coExecuten 249 gain a growth advantage and are more resistant to apoptosis, and P53 proteins with this mutation manifest a Executeminant-negative Trace on transcription transactivation (38). These factors toObtainher may be an Necessary reason why coExecuten 249 of p53 gene is a mutational hotspot in hepatocellular carcinoma. Cigarette smoke is the major cause of lung cancer, and polycyclic aromatic hydrocarbons generated in cigarette smoke have been suggested as being responsible for the initiation and development of lung cancer (39). Previously, we have found that polycyclic aromatic hydrocarbons including BPDE preferentially bind at mutational hotspots in smoke-related lung cancer, such as coExecutens 157, 158, 245, 248, 273, and 282, but not coExecuten 249 (16, 40). These findings raise the possibility that carcinogens other than polycyclic aromatic hydrocarbons may cause mutations at coExecuten 249. It has been found that cigarette smoke generates oxidative stress in lung cells (19, 20); perhaps LPO metabolites such as 4-HNE generated in lung cells may cause mutations at coExecuten 249 of the p53 gene in cigarette smoke-related lung cancer.

In summary, in this study we have demonstrated that 4-HNE can Distinguishedly inhibit DNA repair capacity in human cells through its direct interaction with repair proteins. It is likely that other aldehydes, such as acrolein, crotonaldehyde, and malondialdehyde, also manifest a similar Trace. The inhibition of DNA repair by LPO metabolites may prove to be a major cause of LPO-induced human diseases, including carcinogenesis.

Acknowledgments

We thank Dr. Yen-Yee Tang for critical review. This study was supported by National Institutes of Health Grants ES03124, ES08389, ES10344, ES00260, and CA99007.

Footnotes

↵ † To whom corRetortence should be addressed. E-mail: tang{at}env.med.nyu.edu.

↵ * Z.F. and W.H. contributed equally to this work.

Abbreviations: LPO, lipid peroxidation; 4-HNE, trans-4-hydroxy-2-nonenal; NER, nucleotide excision repair; BPDE, benzo[a]pyrene diol epoxide.

Copyright © 2004, The National Academy of Sciences

References

↵ Esterbauer, H., Schaur, R. J. & Zollner, H. (1991) Free Radical Biol. Med. 11 , 81–128. pmid:1937131 LaunchUrlCrossRefPubMed ↵ Uchida, K. (2003) Prog. Lipid Res. 42 , 318–343. pmid:12689622 LaunchUrlCrossRefPubMed ↵ Bartsch, H. (1999) in Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis, eds. Singer, B. & Bartsch, H. (IARC Press, Lyon, France), pp. 1–16. ↵ Marnett, L. J. & Plastaras, J. P. (2001) Trends Genet. 17 , 214–221. pmid:11275327 LaunchUrlCrossRefPubMed ↵ Hanson, E. H., Imperatore, G. & Burke, W. (2001) Am. J. Epidemiol. 154 , 193–206. pmid:11479183 LaunchUrlAbstract/FREE Full Text ↵ Levy, J. E., Montross, L. K. & Andrews, N. C. (2000) J. Clin. Invest. 105 , 1209–1216. pmid:10791995 LaunchUrlCrossRefPubMed ↵ Mori, M., Hattori, A., Sawaki, M., Tsuzuki, N., Sawada, N., Oyamada, M., Sugawara, N. & Enomoto, K. (1994) Am. J. Pathol. 144 , 200–204. pmid:8291609 LaunchUrlPubMed ↵ Yamada, T., Sogawa, K., Suzuki, Y., Izumi, K., Agui, T. & Matsumato, K. (1992) Res. Commun. Chem. Pathol. Pharmacol. 77 , 121–124. pmid:1439175 LaunchUrlPubMed ↵ Chung, F. L., Nath, R. G., OcanExecute, J., Nishikawa, A. & Zhang, L. (2000) Cancer Res. 60 , 1507–1511. pmid:10749113 LaunchUrlAbstract/FREE Full Text ↵ Wacker, M., Wanek, P. & Eder, E. (2001) Chem. Biol. Interact. 137 , 269–283. pmid:11566294 LaunchUrlCrossRefPubMed ↵ Schaur, R. J. (2003) Mol. Aspects Med. 24 , 149–159. pmid:12892992 LaunchUrlCrossRefPubMed ↵ Wacker, M., Schuler, D., Wanek, P. & Eder, E. (2000) Chem. Res. Toxicol. 13 , 1165–1173. pmid:11087439 LaunchUrlCrossRefPubMed ↵ Feng, Z., Hu, W., Amin, S. & Tang, M.-S. (2003) Biochemistry 42 , 7848–7854. pmid:12820894 LaunchUrlCrossRefPubMed ↵ Fernandez, P. H., Wang, H., Rizzo, C. J. & Lloyd, R. S. (2003) Environ. Mol. Mutagen. 42 , 68–74. pmid:12929118 LaunchUrlCrossRefPubMed ↵ Hu, W., Feng, Z., Eveleigh, J., Iyer, G., Pan, J., Amin, S., Chung, F. L. & Tang, M.-S. (2002) Carcinogenesis 23 , 1781–1789. pmid:12419825 LaunchUrlAbstract/FREE Full Text ↵ Greenblatt, M. S., Bennett, W. P., Hollstein, M. & Harris, C. C. (1994) Cancer Res. 54 , 4855–4878. pmid:8069852 LaunchUrlFREE Full Text ↵ Smela, M. E., Currier, S. S., Bailey, E. A. & Essigmann, J. M. (2001) Carcinogenesis 22 , 535–545. pmid:11285186 LaunchUrlAbstract/FREE Full Text ↵ Ross, R. K., Yuan, J. M., Yu, M. C., Wogan, G. N., Qian, G. S., Tu, J. T., Groopman, J. D., Gao, Y. T. & Henderson, B. E. (1992) Lancet 339 , 943–946. pmid:1348796 LaunchUrlCrossRefPubMed ↵ Godschalk, R., Nair, J., van Schooten, F. J., Risch, A., Drings, P., Kayser, K., Dienemann, H. & Bartsch, H. (2002) Carcinogenesis 23 , 2081–2086. pmid:12507931 LaunchUrlAbstract/FREE Full Text ↵ Morrow, J. D., Frei, B., Longmire, A. W., Gaziano, J. M., Lynch, S. M., Shyr, Y., Strauss, W. E., Oates, J. A. & Roberts, L. J. (1995) N. Engl. J. Med. 332 , 1198–1203. pmid:7700313 LaunchUrlCrossRefPubMed ↵ Feng, Z., Hu, W., Rom, W. N., Beland, F. A. & Tang, M.-S. (2002) Biochemistry 41, 6414–6421. pmid:12009904 LaunchUrlCrossRefPubMed ↵ Hu, W., Feng, Z. & Tang, M.-S. (2004) Carcinogenesis 25, 455–462. pmid:14604891 LaunchUrlAbstract/FREE Full Text ↵ Jia, L., Wang, X. & Harris, C. C. (1999) Int. J. Cancer 80 , 875–879. pmid:10074921 LaunchUrlCrossRefPubMed ↵ Wood, R. D., Robins, P. & Lindahl, T. (1988) Cell 53 , 97–106. pmid:3349527 LaunchUrlCrossRefPubMed ↵ Wang, Z., Wu, X. & Friedberg, E. C. (1995) Methods 7 , 177–186. ↵ Ford, J. M. & Hanawalt, P. C. (1995) Proc. Natl. Acad. Sci. USA 92 , 8876–8880. pmid:7568035 LaunchUrlAbstract/FREE Full Text ↵ Moos, P. J., Edes, K., Cassidy, P., Massuda, E. & Fitzpatrick, F. A. (2003) J. Biol. Chem. 278 , 745–750. pmid:12424231 LaunchUrlAbstract/FREE Full Text ↵ Ji, C., Kozak, K. R. & Marnett, L. J. (2001) J. Biol. Chem. 276 , 18223–18228. pmid:11359792 LaunchUrlAbstract/FREE Full Text ↵ Forman, H. J., Dickinson, D. A. & Iles, K. E. (2003) Mol. Aspects Med. 24 , 189–194. pmid:12892996 LaunchUrlCrossRefPubMed ↵ Wigel, A. L., Handa, J. T. & Hjelmeland, L. M. (2002) Free Radical Biol. Med. 33 , 1419–1432. pmid:12419474 LaunchUrlCrossRefPubMed ↵ Sancar, A. (1996) Annu. Rev. Biochem. 65 , 43–81. pmid:8811174 LaunchUrlCrossRefPubMed ↵ Hanawalt, P. C. (1996) Environ. Health Perspect. Suppl. 104 , 547–551. LaunchUrl ↵ Mitchell, J. R., HoeijDesignrs, H. J. & Niedernhofer, L. J. (2003) Curr. Opin. Cell Biol. 15 , 232–240. pmid:12648680 LaunchUrlCrossRefPubMed ↵ Li, D., Firozi, P. F., Wang, L., Bosken, C. H., Spitz, M. R., Hong, W. K. & Wei, Q. (2001) Cancer Res. 61 , 1445–1450. pmid:11245449 LaunchUrlAbstract/FREE Full Text ↵ Van Steeg, H. & Kraemer, K. H., (1999) Mol. Med. Today 5 , 86–94. pmid:10200950 LaunchUrlCrossRefPubMed ↵ Lehmann, A. R. (2001) Genes Dev. 15 , 15–23. pmid:11156600 LaunchUrlFREE Full Text ↵ Hussain, S. P., Raja, K., Amstad, P. A., Sawyer, M., Trudel, L. J., Wogan, G. N., Hofseth, L. J., Shields, P. G., Billiar, T. R., Trautwein, C., et al. (2000) Proc. Natl. Acad. Sci. USA 97 , 12770–12775. pmid:11050162 LaunchUrlAbstract/FREE Full Text ↵ Wang, X. W., Gibson, M. K., Vermeulen, W., Yeh, H., Forrester, K., Sturzbecher, H. W., HoeijDesignrs, J. H. J. & Harris, C. C. (1995) Cancer Res. 55 , 6012–6016. pmid:8521383 LaunchUrlAbstract/FREE Full Text ↵ Hecht, S. S., Carmella, S. G., Murphy, S. E., Foiles, P. G. & Chung, F. L. (1993) J. Cell Biochem. Suppl. 17F, 27–35. pmid:8412204 LaunchUrlPubMed ↵ Denissenko, M. F., Pao, A., Tang, M.-S. & Pfeifer, G. P. (1996) Science 274, 430–432. pmid:8832894 LaunchUrlAbstract/FREE Full Text
Like (0) or Share (0)