DNA Damage by Endogenous and Exogenous Aldehydes

The present overview describes the formation of deoxyribonucleic acid (DNA) adducts from endogenous and exogenous aldehydes, such as acetaldehyde, acrolein, crotonaldehyde, malonaldehyde, 4-hydroxy-2-nonenal and 2,4-decadienal. Malonaldehyde reacts with 2’-deoxyguanosine, 2’-deoxyadenosine, and 2’-deoxycytidine, yielding cyclic pyrimidopurinone and acyclic adducts. The direct addition of α,β-unsaturated aldehydes to DNA bases yields cyclic substituted propano adducts, such as 1,N-propano-2’-deoxyguanosine. Alternatively, α,βunsaturated aldehydes can be oxidized to reactive epoxides, giving ethano or etheno derivatives upon reaction with DNA. In addition, information on highly sensitive techniques, employed for the in vivo detection and quantification of DNA-aldehyde adducts, is also provided. Some of these DNA-aldehyde lesions have been shown to be highly mutagenic. In fact, lipid peroxidation and exogenous aldehyde exposure could potentially account for the observed carcinogenicity of urban air pollution and cigarette smoke exposure.


Introduction
The integrity and stability of genetic information is crucial for maintaining life.However, deoxyribonucleic acid (DNA) is not inert and has numerous sites for chemical interaction.For example, various types of DNA lesions, resulting from attack on nitrogenous bases, 2'-deoxyribose residues and phosphodiester bonds, have been reported. 1It is estimated that the DNA in a single cell can undergo up to one million changes per day. 2 In fact, various sources including UV radiation, ionizing radiation, as well as genotoxic agents present in the air, food and cigarette smoke have been shown to modify DNA.
Aldehydes are known to react with and modify DNA.These compounds are widespread in the environment, and are present in foods, beverages, cigarette smoke, and are also formed through the combustion of wood, coal, alcohol and diesel fuels. 3Exogenous aldehydes, such as acrolein, 2,4-nonadienal and 2-pentenal, are also formed during the cooking of fats, oils, and sugars. 4Aldehydes are produced endogenously, primarily, by the lipid peroxidation, which produces a large number of reactive aldehydes.Many of these aldehydes react with biomolecules such as DNA, proteins and amino acids, ultimately resulting in a variety of diseases and cytotoxic effects, and contributing to the aging process (Figure 1). 5,6ldehydes are capable of modifying DNA and yielding promutagenic lesions, which may, at least partially, account for the observed mutagenic and carcinogenic effects associated with the lipid peroxidation process and urban air

DNA Damage by Endogenous and Exogenous Aldehydes
Marisa H. G. Medeiros * ,a

The Lipid Peroxidation Process
Lipid peroxidation is an important redox stress event that has been linked to the development of several pathologies such as cancer, as well as neurodegenerative and inflammatory diseases.The polyunsaturated fatty acid side chains of membrane phospholipids undergo enzymatic and non-enzymatic oxidation events, which are known to generate a complex mixture of phospholipid products, including hydroperoxides, that can decompose to electrophilic derivatives, such as aldehydes and epoxyaldehydes (Figure 2). 8he lipid peroxidation initiation involves abstraction of a bis-allylic hydrogen atom from ω-3 and ω-6 unsaturated fatty acids by an oxidant.This is followed by radical chain reaction, that leads to the formation of lipid hydroperoxides, intramolecular rearrangement and chain-breaking reactions. 9he decomposition of lipid hydroperoxides is important because, in addition to generating radicals that propagate the lipid peroxidation process, it also generates non-radical products (Figure 2).These by-products (alkanes, alkenes, aldehydes, ketones, hydroxy acids) are more stable than the free radicals that initiated the process and the lipid radicals formed during the propagation phase.Consequently, these non-radical products are more toxic because they can reach points distant from where they were formed. 8Biological systems contain a mixture of different polyunsaturated fatty acids with varying degrees of unsaturation.As a consequence, lipid peroxidation generates a mixture of lipid hydroperoxides, which can ultimately produce a variety of different aldehydes and radical species. 10eactive aldehydes including 4-hydroxy-trans-2nonenal (HNE), 2,4-decadienal (DDE), malondialdehyde (MDA), 9,11 [11][12][13] are all formed as secondary lipid peroxidation products (Figure 3).
A growing number of studies have shown that aldehydes interact directly with DNA and cause genetic damage, or are metabolized to epoxides, compounds known to be alkylating agents of DNA with high mutagenic activity. 14,15The most extensively studied final products are the aldehydes, MDA and HNE.MDA is highly toxic to cells and has been considered a marker for lipid peroxidation. 16HNE has been extensively studied because of its high reactivity with a large number of biomolecules. 17dditionally, it has many cytotoxic effects, such as the inhibition of enzyme activity, protein synthesis, DNA and ribonucleic acid (RNA) synthesis, as well as the induction of heat shock proteins and blockage of cell proliferation. 17,18NE has also been shown to possess genotoxic 19,20 and mutagenic properties. 20This aldehyde was also present in oil samples collected during the Spanish oil syndrome. 11It is well established that HNE is a strong electrophile which reacts preferentially with compounds having thiol groups (cysteine, glutathione, SH-containing proteins), and is less reactive with compounds containing amino groups. 4On the other hand, it has been reported that another secondary lipid peroxidation product, the 4-oxo-2-nonenal, is more reactive with sulfhydryl groups than HNE and, is more reactive with DNA than other aldehydes. 21awai et al. 22 observed an increase in aldehydes in the liver of mice intraperitoneally injected with bromobenzene, an experimental animal model for lipid peroxidation; however, the authors pointed out that the types of aldehydes formed depends on which polyunsaturated fatty acids are present in the membrane.Therefore, these results indicate the importance of monitoring not only a specific aldehyde or the total amount of aldehydes, as in the TBARS (thiobarbituric acid reactive substances) assay, but also all individual aldehydes.

Endogenous and Exogenous Acetaldehyde
Acetaldehyde is commonly found in foods, beverages, cigarette smoke and fuel combustion, and, consequently, widespread throughout the environment. 7Acetaldehyde is a mutagenic and carcinogenic compound capable of inducing mutations (G → A transitions and G → T transversions), and sister chromatid exchanges in rodent spinal cord cells, squamous epithelium and cultured human lymphocytes. 7,56,57cetaldehyde is carcinogenic to rats and hamsters, inducing respiratory tract tumors after inhalation. 58It is formed endogenously as a product of ethanol oxidation and has been found in the liver and saliva after ethanol ingestion. 59,60cetaldehyde is also produced in small amounts during threonine catabolism. 61It is noteworthy that populations deficient in aldehyde dehydrogenase have a higher risk of developing esophageal cancer associated with alcohol consumption, when compared to populations with fully active enzyme. 62,63The formation of DNA adducts has been considered to be a key factor in the acetaldehyde toxicity mechanism. 64This compound has also been shown to react with DNA.For example, when acetaldehyde reacts with 2'-deoxyguanosine (dGuo), N 2 -ethylidene-2'-deoxyguanosine (N 2 -ethyldGuo), and an unstable Schiff base are formed. 65The N 2 -ethyldGuo can then react with a second molecule of acetaldehyde forming the (6S, 8S) and (6R, 8R) diastereomers of 1,N 2 -propanodGuo adducts (Figure 6). 66he formation of 1,N 2 -propanodGuo, as a two-step reaction, was unequivocally demonstrated by treating cells with [ 13 C 2 ]-acetaldehyde and detecting the labeled adduct with HPLC-ESI-MS/MS (Figure 7). 67 h e r e d u c e d f o r m o f N 2 -e t h y l i d e n e d G u o was also quantified in DNA from cells treated with [ 13 C 2 ]-acetaldehyde, and it was observed that these adducts were present at levels comparable to those of 1,N 2 -propanodGuo.A similar result was also reported in cells exposed to high concentrations of acetaldehyde. 68It was suggested that the formation of 1,N 2 -propanodGuo was unfavorable at low acetaldehyde concentrations, and that N 2 -ethylidenedGuo formed at higher rates.Therefore N 2 -ethylidenedGuo should be the main adduct formed under typical human exposure conditions. 67olyamines and histones catalyze the formation of 1,N 2 -propanodGuo.The reaction of crotonaldehyde with dGuo, as well as with DNA forms 1,N 2 -propanodGuo.In addition to being an industrial pollutant, crotonaldehyde is also produced by the lipid peroxidation and is a metabolite of N-nitrosopyrrolidine. 69 In DNA, the 1,N 2 -propanodGuo adduct exists in equilibrium between open and closed forms.The open form is favored in double-stranded DNA, whereas the closed form predominates in single-stranded DNA. 70terestingly, high levels of 1,N 2 -propanodGuo (20.8 fmol of 1,N 2 -propanodGuo per mg creatinine) were found in the urine of residents living in a polluted region of São Paulo City, in Brazil, when compared with urine from residents of an unpolluted Brazilian city (São João da Boa Vista, São Paulo State) (7.9 fmol of 1,N 2 -propanodGuo per mg creatinine). 71ir pollution has been associated with increased mortality among various age groups and responsible for causing several adverse health effects. 72The investigation of the mutagenicity of organic solvent extracts of PM 10 (particulate matter with aerodynamic diameters of less than 10 μm) collected from SP was performed by de Martinis et al., 73 and found that the most mutagenic extract  fractions contained aldehydes, ketones, carboxylic acids and quinolines.Recently, it was shown that mice exposed daily to PM 2.5 (particulate matter with aerodynamic diameters of less than 2.5 μm), for 3 months, at a concentration that mimics a 24 h exposure to the mean concentration found in ambient air presented, after 3 months, increased levels of DNA lesions consistent with oxidative stress in the lungs, liver and kidney.Additionally, it has been proposed that genetic and epigenetic alterations induced by pollutants may increase the chance of cancer development. 74tudies with inhaled acetaldehyde in animal models, have been performed since 1900, when the anesthetic properties of aldehyde in animals was shown by Lewin. 75ubsequent, studies have demonstrated the carcinogenic effects of acetaldehyde in different animal models breathing very high concentrations of acetaldehyde. 76Furthermore, rats chronically exposed (6 h per day, five days a week for 52 weeks) to acetaldehyde concentrations of greater than 400 ppm displayed degeneration of the olfactory epithelium.At higher acetaldehyde concentrations, effects ranging from hyper-and metaplasia of olfactory epithelium cells to the development of cancer (squamous cell and adenocarcinomas) were reported. 58,77,78he unequivocal formation of labeled 1,N 2 -propanodGuo in DNA was also verified, by micro-HPLC-MS/MS, in the lung and brain tissues of rats that inhaled environmentally relevant doses of [ 13 C 2 ]-acetaldehyde.Additionally, the structure of the products was confirmed by nanoflow high-performance liquid chromatography-electrospray ionization high resolution tandem mass spectrometry in the positive mode 3 analyses.Together these results indicated that the levels of the 1,N 2 -propanodGuo adduct could be potentially utilized as a biomarker of acetaldehyde and crotonaldehyde exposure, and that monitoring these levels could protect the exposed population against the related adverse effects of this chemical. 79

DNA-Adducts Quantification in vivo
Efforts have been made to determine the levels of exocyclic DNA adducts generated by exposure to electrophilic molecules, from both exogenous and endogenous sources.Indeed, in vivo studies showed that endogenous sources generated approximately 0-20 lesions per 10 8 nucleotides. 12,23,80Due to the extremely low levels of these adducts in biological systems, ultrasensitive methods are required for their detection and quantification. 5Examples of such techniques include immunoassays, 32 P-postlabeling, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS).Among these ultrasensitive methods, LC-MS/ MS is considered to be the most precise and specific method for quantifying exocyclic DNA adducts.The confidence level of this method can be further improved by including an isotopically labeled internal standard prior to DNA hydrolysis, which allows for the correction of any analyte loss during the procedure.In fact, the current mass spectrometry technology is capable of quantifying exocyclic DNA adducts in the range of 10-18 amol. 79

Mechanism of in vivo Aldehyde Detoxification
Endogenous and exogenous aldehydes are metabolized to less toxic products by oxidation / reduction (phase I) or conjugation (phase II) mechanisms.The phase I mechanism employs enzymes such as glutathione S-transferase (GST), aldehyde dehydrogenase (ALDH), aldo-keto reductase (AKR), cytochrome P450 and alcohol dehydrogenase, which are directly involved in aldehyde detoxification. 81,82A wellknown phase II mechanism for aldehyde detoxification in cells involves the conjugation of these aldehydes with glutathione (GSH), which yields Michael adducts.Additionally, endogenous histidine-containing dipeptides such as carnosine (β-alanyl-L-histidine, CAR), homocarnosine (γ-amino-butyryl-histidine) and anserine (β-alanyl-L-1-methylhistidine) have been shown to detoxify aldehydes through phase II mechanisms. 83Moreover, carnosine has been found at high concentrations in skeletal muscle, as well as in the central nervous system. 846][87] Interestingly, it was shown that administering carnosine (2 g per day), to overweight individuals resulted in a significant increase in the amount of carnosine-acrolein adducts excreted in the urine. 88In fact, our group elucidated the structure of a 3-methylpyridinium carnosine, resulting from the reaction between carnosine and acrolein, and simultaneously quantified carnosine-aldehyde adducts in human urine. 89Recently, carnosine-aldehyde adducts were quantified by LC-MS/MS in human skeletal muscle samples after acute exercise, before and after β-alanine supplementation. 90This study demonstrated that there was a significant increase in post-exercise carnosineacrolein levels following β-alanine supplementation, whereas neither exercise or supplementation alone increased the formation of this adduct.

Conclusions
A variety of chemicals have been shown to alkylate DNA bases.In fact, some of these modifications are formed in human tissues after exposure to reactive aldehydes, resulting in the formation of exocyclic adducts.DNA adducts produced by exogenous and endogenous aldehydes are currently becoming recognized as potential tools for studying a variety of human diseases, as well as the effects of air pollution exposure.However, a systematic investigation of these lesions is necessary for identifying which type of lesion is the most critical in each situation.Ultra-sensitive mass spectrometry techniques have been utilized for understanding the mechanisms involved in the generation of these adducts, as well as for the identification of novel biomarkers associated with these modifications.Advances in mass spectrometry technology are improving exocyclic DNA adduct detection, especially in the blood and urine, which may provide novel noninvasive clinically applicable assays.

Figure 1 .
Figure 1.Biological consequences of DNA damage induced by exogenous and endogenous aldehydes.

Figure 4 .
Figure 4. Structure of exocyclic DNA adducts formed by the reaction of DNA with aldehyde.