Malondialdehyde,a product of lipid peroxidation,is mutagenic in human cells

Malondialdehyde (MDA) is an endogenous genotoxic product of enzymatic and oxygen radical-induced lipid peroxidation whose adducts are known to exist in DNA isolated from healthy human beings. To evaluate the mutagenic potential of MDA in human cells, we reacted MDA with pSP189 shuttle vector DNA and then transfected them into human fibroblasts for replication. MDA induced up to a 15-fold increase in mutation frequency in the supF reporter gene compared with untreated DNA. Sequence analysis revealed that the majority of MDA-induced mutations occurred at GC base pairs. The most frequent mutations were large insertions and deletions, but base pair substitutions were also detected. MDA-induced mutations were completely abolished when the adducted shuttle vector was replicated in cells lacking nucleotide excision repair. MDA induction of large deletions and the apparent requirement for nucleotide excision repair suggested the possible involvement of a DNA interstrand cross-link as a premutagenic lesion. Indeed, MDA formed interstrand cross-links in duplex plasmids and oligonucleotides. Substrates containing the sequence 5'-d(CG) were preferentially cross-linked, consistent with the observation of base pair substitutions in 5'-d(CG) sites in the MDA-induced mutation spectrum. These experiments provide biological and biochemical evidence for the existence of MDA-induced DNA interstrand cross-links that could result from endogenous oxidative stress and likely have potent biological effects.     

Measurement of Lipid Peroxidation

Lipid peroxidation results in the formation of conjugated dienes, lipid hydroperoxides and degradation products such as alkanes, aldehydes and isoprostanes. The approach to the quantitative assessment of lipid peroxidation depends on whether the samples involve complex biological material obtained in vivo, or whether the samples involve relatively simple mixtures obtained in vituo. Samples obtained in vivo contain a large number of products which themselves may undergo metabolism. The measurement of conjugated diene formation is generally applied as a dynamic quantitation e.g. during the oxidation of LDL, and is not generally applied to samples obtained in vivo. Lipid hydroperoxides readily decompose, but can be measured directly and indirectly by a variety of techniques. The measurement of MDA by the TBAR assay is non-specific, and is generally poor when applied to biological samples. More recent assays based on the measurement of MDA or HNE-lysine adducts are likely to be more applicable to biological samples, since adducts of these reactive aldehydes are relatively stable. The discovery of the isoprostanes as lipid peroxidation products which can be measured by gas chromatography mass spectrometry or immunoassay has opened a new avenue by which to quantify lipid peroxidation in vivo, and will be discussed in detail.     

Determination of hemoglobin adducts from aldehydes formed during lipid peroxidation in vitro.

This study was performed in order to investigate the formation of hemoglobin (Hb) adducts from some aldehydes, known to be formed during the peroxidation of polyunsaturated fatty acids. Hb was reacted in vitro with aldehydes followed by reduction of hemolysate with sodium borohydride and isolation of globin. Using the N-alkyl Edman method, globin samples were derivatized with pentafluorophenyl isothiocyanate for specific splitting off of N-substituted N-terminal valines. Adducts formed were then characterized by gas chromatography mass spectrometry using different ionization techniques. It was shown that adducts from saturated aldehydes were Schiff bases and those from alpha,beta-unsaturated aldehydes were mixtures of Schiff bases and 1,4-addition products. Aldehyde adducts were also measured in Hb from erythrocytes induced for lipid peroxidation. Incubation of the erythrocytes in presence of arachidonic acid led to increased levels of adducts.     

DNA damage induced by endogenous aldehydes: current state of knowledge

DNA damage plays a major role in various pathophysiological conditions including carcinogenesis, aging, inflammation, diabetes and neurodegenerative diseases. Oxidative stress and cell processes such as lipid peroxidation and glycation induce the formation of highly reactive endogenous aldehydes that react directly with DNA, form aldehyde-derived DNA adducts and lead to DNA damage. In occasion of persistent conditions that influence the formation and accumulation of aldehyde-derived DNA adducts the resulting unrepaired DNA damage causes deregulation of cell homeostasis and thus significantly contributes to disease phenotype. Some of the most highly reactive aldehydes produced endogenously are 4-hydroxy-2-nonenal, malondialdehyde, acrolein, crotonaldehyde and methylglyoxal. The mutagenic and carcinogenic effects associated with the elevated levels of these reactive aldehydes, especially, under conditions of stress, are attributed to their capability of causing directly modification of DNA bases or yielding promutagenic exocyclic adducts. In this review, we discuss the current knowledge on DNA damage induced by endogenously produced reactive aldehydes in relation to the pathophysiology of human diseases.     

Binding of annexin V to membrane products of lipid peroxidation

There is increasing evidence that endogenously generated aldehydes formed as a result of lipid peroxidation are involved in the pathophysiological effects associated with oxidative stress in cells and tissues. Malondialdehyde (MDA), a major product of lipid peroxidation, can modify amines present on the cell surface and thereby introduce negative charges that can affect the interfacial ionic layer. We show that lipid peroxidation of RBC generates MDA adducts that, similar to phosphatidylserine (PS), bind annexin V in a Ca(2+)-dependent manner. Like PS, these adducts also promote the "PS-dependent" prothrombinase assays, albeit to lower levels. These results indicate that annexin V binding cannot be used as an exclusive indicator of cell surface PS and raise the possibility that some phenomenon attributed to PS may, in fact, also involve aldehyde-lipid adducts.     

Enzymology of repair of etheno-adducts

Etheno(epsilon)-adducts such as 1,N(6)-ethenoadenine (epsilon A), 3,N(4)-ethenocytosine (epsilon C), N(2),3-ethenoguanine (N(2),3-epsilon G), and 1,N(2)-ethenoguanine (1,N(2)-epsilon G) are produced in cellular DNA by two independent pathways: (i) by reaction with oxidised metabolites of vinyl chloride, 2-chloroacetaldehyde and 2-chloroethylene oxide; (ii) by endogenous processes through the interaction of lipid peroxidation (LPO)-derived aldehydes and hydroxyalkenals. They have been found in DNA isolated from human and rodent tissues. However, the levels of adducts were significantly increased by cancer risk factors contributing to lipid peroxidation and oxidative stress.The highly mutagenic and genotoxic properties of epsilon-adducts have been established in vitro by analysing steady-state kinetics of primer extension assays and in vivo by site-specific mutagenesis in mammalian cells. Therefore, the repair processes eliminating exocyclic adducts from DNA should play a crucial role in maintaining the stability of genetic information. The epsilon-adducts are eliminated by the base excision repair (BER) pathway, with DNA glycosylases being the key enzymes of this pathway. They remove epsilon-adducts from DNA by hydrolysing the N-glycosidic bond between the damaged base and deoxyribose, leaving an abasic site in DNA. The ethenobase-DNA glycosylases have been identified and their enzymatic properties described. They are specific for a given epsilon-base although they can also excise different types of modified bases, such as alkylated purines, hypoxanthine and uracil. The fact that ethenoadducts are recognised and excised with high efficiency by various DNA glycosylases in vitro suggests that these enzymes may be responsible for repair of these mutagenic lesions in vivo, and thus constitute important contributors to genetic stability.     

Exocyclic malondialdehyde and aromatic DNA adducts in larynx tissues

Cigarette smoking and alcohol consumption, known to cause free radical generation and lipid peroxidation, are established risk factors for larynx cancer. Malondialdehyde (MDA) is a naturally occurring product of lipid peroxidation, capable of interacting with DNA to form exocyclic MDA-DNA adducts. In the present study, we investigated if the production of MDA-DNA adducts was increased in larynx cancer patients with respect to controls using (32)P-DNA postlabeling techniques. Moreover, we examined the potential effects of cigarette smoking and alcohol consumption on endogenous DNA adducts. We then analyzed the same set of larynx tissues for the presence of (32)P-postlabeled aromatic DNA adducts to determine more about the levels and types of adducts formed in the larynx. We observed that cancer patients tended to have increased levels of MDA and aromatic DNA adducts with respect to controls. In addition, smoking and alcohol were found to influence the formation of endogenous adducts in the larynx tissues. Finally, the amounts of endogenous adducts were found to be comparable to those observed for aromatic DNA adducts in the same set of larynx tissues. These findings imply that endogenous lesions, if not repaired, may contribute to larynx cancer development.     

Characterization of bis(levuglandinyl) urea derivatives as products of the reaction between prostaglandin H2 and arginine

Levuglandins are gamma-keto aldehydes formed by rearrangement of prostaglandin (PG) H(2) in aqueous solution. Levuglandins are highly reactive with primary amines. We had previously characterized adducts formed after reaction of levuglandin E(2) (LGE(2)) or PGH(2) with lysine. In this study, we assessed whether reaction of PGH(2) with arginine yielded covalent adducts. Using N(alpha)-acetylarginine and both PGH(2) and synthetic LGE(2), we discovered a novel series of levuglandinyl adducts derived from reaction of two levuglandin moieties with the guanidino group of arginine. Subsequent spontaneous hydrolysis of the adducted amino acid yields bis(levuglandinyl) urea and the corresponding ornithine residue. Using liquid chromatography tandem mass spectrometry, we characterized the molecular structure of these novel adducts and demonstrated their formation after coincubation of PGH(2) with synthetic peptides and proteins. The soluble characteristic of these molecules provides a potential strategy for development of biological markers of lipid modification of proteins following cyclooxygenase activity or lipid peroxidation.     

Powerful mutagenicity of a bipyridylium herbicide in a nitrogen-fixing blue-green alga Nostoc muscorum

Malondialdehyde (MDA), an in vivo metabolite of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in Salmonella typhimurium. It is a reactive electrophile that can form interstrand cross-links in DNA. To explore the possibility that MDA-induced interstrand cross-links are the pre-mutagenic lesion, we have quantitated the ability of highly purified preparations of MDA to form interstrand cross-links when reacted with linear plasmid DNA. At physiological temperature and pH, MDA did not form DNA cross-links as determined by DNA denaturation followed by agarose gel electrophoresis. DNA cross-links were formed, however, when incubations with MDA were carried out at either pH 4.2 or temperatures exceeding 60 degrees. alpha-Methylmalondialdehyde (CH3MDA) was found to cross-link DNA more efficiently than MDA, but was not mutagenic in any tester strain of Salmonella. MDA polymers, formed by acid incubation of MDA, also were capable of inducing cross-links. However, an inverse relationship was observed between mutagenicity and extent of polymerization. The pattern of mutagenic response for MDA in different strains of Salmonella was compared with mitomycin C, an established mutagenic cross-linking agent. Error-prone repair and a UvrB+ phenotype, which are needed for the induction of mutations by mitomycin C, were not required for MDA mutagenesis. These findings, taken together, dissociate the mutagenicity of MDA from its ability to form interstrand cross-links with DNA.     

Covalent modifications of aminophospholipids by 4-hydroxynonenal

Lipid oxidation is implicated in a wide range of pathophysiological disorders, which leads to reactive compounds such as aldehydes. Among them 4-hydroxynonenal (4-HNE) reacts strongly with the NH₂ groups of amino acids and forms mainly Michael adducts and minor Schiff-base adducts. Such reactions occur also with compounds containing thiol groups. No data are available describing 4-HNE interactions with amino-phospholipids. To investigate such a possibility, 4-HNE was incubated with either phosphatidylethanolamine (PE) or phosphatidylserine (PS) in an aqueous-organic biphasic system and the resulting products were identified by liquid chromatography-mass spectrometry (LC-MS). Our study points out the potential capacity of 4-HNE to react with phospholipids containing amino groups and particularly PE. The main resulting compounds found were a Michael adduct plus a minor Schiff base adduct, which was partly cyclized as a pyrrole derivative via a loss of water. Its stabilization as a pyrrole derivative allows to differentiate 4-HNE from the other aldehydes generated via lipid oxidation (e.g., malondialdehyde, 2-nonenal) that lack the 4-hydroxyl group. Their formation seems not to be affected when the pH varies from 6.5 to 8.5. Surprisingly, PS reacted poorly producing only a small amount of Michael adduct, the Schiff-base adduct being nondetectable. We conclude that such adducts, if they are formed in cell membranes, could alter the phospholipase-dependent cell signaling.     

Urinary aldehydes as indicators of lipid peroxidation in vivo

The excretion of malondialdehyde (MDA), lipophilic aldehydes and related carbonyl compounds in rat and human urine was investigated. MDA was found to be excreted mainly in the form of two adducts with lysine, indicating that its predominant reaction in vivo is with the lysine residues of proteins. Adducts with the phospholipid bases serine and ethanolamine and the nucleic acid bases guanine and deoxyguanosine also were found. Except for the adduct with deoxyguanosine (dG-MDA), the excretion of these compounds increased with peroxidative stress imposed in the form of vitamin E deficiency or the administration of iron or carbon tetrachloride. Marked differences in the concentration of dG-MDA in different tissues were correlated with their content of fatty acids having three or more double bonds, the putative source of MDA. Fourteen nonpolar and eleven polar lipophilic aldehydes and other carbonyl compounds were identified as their 2,4-diphenylhydrazine derivatives in rat urine. The excretion of five nonpolar and nine polar compounds was increased under conditions of peroxidative stress. The profile of lipophilic aldehydes obtained for human urine resembled that for rat urine. Except for a reported 4-hydroxynon-2-enal conjugate with mercapturic acid, the conjugated forms of the lipophilic aldehydes excreted in urine remain unidentified. Aldehyde excretion is influenced by numerous factors that affect the formation of lipid peroxides in vivo such as energy status, physical activity and environmental temperature, as well as by wide variations in the intake of peroxides in the diet. Consequently, urinalysis for aldehydic products of lipid peroxidation is an unreliable indicator of the general state of peroxidative stress in vivo.     

Lipid peroxidation-induced DNA damage in cancer-prone inflammatory diseases: a review of published adduct types and levels in humans

Persistent oxidative stress and excess lipid peroxidation (LPO), induced by inflammatory processes, impaired metal storage, and/or dietary imbalance, cause accumulations and massive DNA damage. This massive DNA damage, along with deregulation of cell homeostasis, leads to malignant diseases. Reactive aldehydes produced by LPO, such as 4-hydroxy-2-nonenal, malondialdehyde, acrolein, and crotonaldehyde, react directly with DNA bases or generate bifunctional intermediates which form exocyclic DNA adducts. Modification of DNA bases by these electrophiles, yielding promutagenic exocyclic adducts, is thought to contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. Ultrasensitive detection methods have facilitated studies of the concentrations of promutagenic DNA adducts in human tissues, white blood cells, and urine, where they are excreted as modified nucleosides and bases. Thus, immunoaffinity-(32)P-postlabeling, high-performance liquid chromatography-electrochemical detection, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, immunoslotblot assay, and immunohistochemistry have made it possible to detect background concentrations of adducts arising from endogenous LPO products in vivo and studies of their role in carcinogenesis. These background adduct levels in asymptomatic human tissues occur in the order of 1 adduct/10(8) and in organs affected by cancer-prone inflammatory diseases these can be 1 or 2 orders of magnitude higher. In this review, we critically discuss the accuracy of the available methods and their validation and summarize studies in which measurement of exocyclic adducts suggested new mechanisms of cancer causation, providing potential biomarkers for cancer risk assessment in humans with cancer-prone diseases.     

Lipid aldehyde-mediated cross-linking of apolipoprotein B-100 inhibits secretion from HepG2 cells

Hepatic oxidative stress and lipid peroxidation are common features of several prevalent disease states, including alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), a common component of the metabolic syndrome. These conditions are characterized in part by excessive accumulation of lipids within hepatocytes, which can lead to autocatalytic degradation of cellular lipids giving rise to electrophilic end products of lipid peroxidation. The pathobiology of reactive lipid aldehydes remains poorly understood. We therefore sought to investigate the effects of 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) on the transport and secretion of very low-density lipoprotein using HepG2 cells as a model hepatocyte system. Physiologically relevant concentrations of 4-HNE and 4-ONE rapidly disrupted cellular microtubules in a concentration-dependent manner. Interestingly, 4-ONE reduced apolipoprotein B-100 (ApoB) secretion while 4-HNE did not significantly impair secretion. Both 4-HNE and 4-ONE formed adducts with ApoB protein, but 4-HNE adducts were detectable as mono-adducts, while 4-ONE adducts were present as protein–protein cross-links. These results demonstrate that reactive aldehydes generated by lipid peroxidation can differ in their biological effects, and that these differences can be mechanistically explained by the structures of the protein adducts formed.     

4-oxo-2-hexenal, a mutagen formed by omega-3 fat peroxidation: occurrence, detection and adduct formation

The purpose of this review is to summarize our recent studies of a novel mutagen, 4-oxo-2-hexenal. To identify the mutagens formed in a model reaction of lipid peroxidation, linolenic acid methyl ester and hemin were reacted with dG. A 4-oxo-2-hexenal-dG adduct (dG*) was identified in the model reaction mixture. The 4-oxo-2-hexenal (4-OHE) showed mutagenic activity in the Salmonella typhimurium strains TA100 and TA104. 4-OHE reacts with DNA to form dG, dC, and 5-methyl-dC(5-Me-dC)-adducts (dG*, dC*, 5-Me-dC*) in vitro. After 4-OHE was orally administered to mice, these adducts were detected in esophageal, stomach and intestinal DNA by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). We also confirmed the formation of 4-OHE during the heat processing of edible vegetable oil, and during cooking. It was present at an especially high concentration in broiled saury. 4-OHE is probably generated by the oxidation of omega-3 fats. These results provide a warning to humans, who may be exposed to this mutagen. Since 4-OHE induces DNA adduct formation in experimental animal organs, further studies on the carcinogenicity of 4-OHE and the detection of 4-OHE-DNA adducts in human tissue will be required.     

Identification of spin trapped carbon-centered radicals in soybean lipoxygenase-dependent peroxidations of omega-3 polyunsaturated fatty acids by LC/ESR,LC/MS,and tandem MS

With the combined techniques of on-line liquid chromatography/electron spin resonance (LC/ESR) and on-line liquid chromatography/mass spectrometry (LC/MS), we have previously characterized all classes of lipid-derived carbon-centered radicals (*Ld) formed from omega-6 polyunsaturated fatty acids (PUFAs: linoleic acid and arachidonic acid). In the present study, the carbon-centered radicals formed from two omega-3 PUFAs (linolenic acid and docosahexaenoic acid) resulting from their reactions with soybean lipoxygenase in the presence of alpha-[4-pyridyl 1-oxide]-N-tert-butylnitrone (POBN) were investigated using the combination of LC/ESR and LC/MS techniques. A total of 16 POBN trapped carbon-centered radicals formed from the peroxidation of linolenic acid and 11 formed from the peroxidation of docosahexaenoic acid were detected by LC/ESR, identified by LC/MS, and structurally confirmed by tandem mass analysis (MS/MS). The on-line ESR chromatograms and MS chromatograms obtained from two omega-3 PUFAs closely resembled each other not only because the four major beta-scission products, including an ethyl radical and three isomeric pentenyl radicals, were formed from each PUFA, but also because isomeric POBN adducts of lipid dihydroxyallylic radicals from both PUFAs had almost identical chromatographic retention times.     

In vitro evolution of RNA aptamers recognizing carcinogenic aromatic amines

The modification of cellular DNA by environmental substances is thought to be a crucial event in chemical induced carcinogenesis. Among the environmental carcinogens, aromatic amines are known for the fact that they can induce several types of cancers through the formation of so-called DNA adducts. We took advantage of the potential of the SELEX method to select for highly specific RNA ligands that recognize specific genotoxic aromatic amines. The aromatic amine 4,4'-methylenedianiline (MDA) was used as a target. Following in vitro selection, we obtained specific MDA-binding RNA molecules based on an affinity chromatography assay. These results open the possibility of using the SELEX technique to generate RNA molecules as diagnostic tools for the detection of DNA damaging compounds and ultimately DNA adducts.     

Prevention of cancer and other chronic diseases worldwide based on sound mechanisms

The transformation of normal cells by DNA reactive, genotoxic carcinogens and the growth promotion and development of mutated cells by enhancing factors is involved in the overall basic mechanism of cancer induction. Thus, discrimination between genotoxic carcinogens and nongenotoxic chemicals is essential. The dose-response curves, reversibility, and organ-and species specificity are distinct. Genotoxic carcinogens are mutagenic, form DNA adducts, induce DNA repair, and form hydroxy radicals and inappropriate peroxidation reactions that antioxidants such as those in vegetables, fruits, and tea can decrease. In contrast, promoters do not form DNA adducts, but raise cell duplication rates, among other attributes. In the USA, about 35% of known cancers are associated with tobacco use and about 55% with inappropriate nutritional habits. Cancer induction can be decreased by avoiding the formation of carcinogens, reducing their metabolic activation, or increasing their detoxification. Excessive dietary salt, and heterocyclic arylamines formed in cooking of meats or fish, and high intake of 40% of calories in fats are health risks, but vegetables, fruits, tea, soy products, and fibers are protective. We review nutritional factors involved in cancer and chronic disease causation and prevention.     

Endogenous genotoxic agents and processes as a basis of spontaneous carcinogenesis

A list of endogenous DNA-damaging agents and processes is given. Endogenous electrophiles are found with the cosubstrates of physiological transfer reactions (S-adenosylmethionine for methylation, ATP for phosphorylation, NAD+ for ADP-ribosylation, acetyl CoA for acetylation). Aldehyde groups (glyceraldehyde-3-phosphate, formaldehyde, open forms of reducing sugars, degradation products of peroxidation) or alkylating degradation products derived from endogenous nitroso compounds represent additional possibilities. Radical-forming reactions include leakage of the superoxide anion radical from terminal cytochromes and redox cycles, hydroxyl radical formation by the Fenton reaction from endogenous hydrogen peroxide, and the formation of lipid peroxides. Genetic instability by spontaneous deaminations and depurinations as well as replicative instability by tautomer errors and in the presence of mutagenic metal ions represent a third important class of endogenous genotoxic processes. The postulated endogenous genotoxicity could form the mechanistic basis for what is called 'spontaneous' tumor incidence and explain the possibility of an increased tumor incidence after treatment of animals with non-genotoxic compounds exhibiting tumor-promoting activity only. Individual differences are expected to be seen also with endogenous DNA damage. The presence of endogenous DNA damage implies that exogenous DNA-carcinogen adducts give rise to an incremental damage which is expected to be proportional to the carcinogen dose at lowest levels. An increased tumor risk due to exposure to exogenous genotoxic carcinogens could therefore be assessed in terms of the background DNA damage, for instance in multiples of the mean level or of the interindividual variability in a population.     

Identification of N alpha-acetyl-epsilon-(2-propenal)lysine as a urinary metabolite of malondialdehyde

Although orally administered malondialdehyde (MDA), a reactive hepatotoxic and mutagenic product of lipid peroxidation, is extensively metabolized to CO2, a portion is excreted in the urine in acid labile "bound" forms. Since much of the MDA in the diet is apparently bound to protein, the metabolism of protein-bound MDA was investigated. MDA was reacted with serum albumin and fed to rats. A urinary metabolite was detected which was shown to be identical to a metabolite of the lysine-MDA enaminal N epsilon-(2-propenal)lysine. After isolation by ion exchange and high performance liquid chromatography the metabolite was identified using high field nuclear magnetic resonance spectroscopy and fast atom bombardment-mass spectroscopy as N alpha-acetyl-epsilon-(2-propenal)lysine. This compound also was a major urinary metabolite of the Na enol salt of MDA administered by stomach intubation, and was excreted in increased amounts by rats fed a diet containing a highly peroxidizable oil (cod liver oil). It was also detected in the urine of fasted animals after injection with NaMDA, indicating that it is formed as a product of lipid peroxidation in vivo as well as of peroxidation of dietary lipids.