Endogenus and Exogenus DNA damaging agents

DNA damages are repaired by many overlapping repair pathways to remove damaged DNA and it also determines cell fate. Nucleotide excision repair pathways repair oxidative base damage and base excision repair pathway removes helical distortions in DNA structure, damages caused by ultra violet radiations and other exogenous oxidative damages. Along with these pathways, DNA Mismatch repair and DNA strand break repair pathways also important in repairing damaged DNA .

Endogenus and Exogenus DNA damaging agents:

Lipid peroxidation

Phospholipid residues of fatty acids are much sensitive to oxidation. Unsaturated fatty acid yields lipid hydroperoxides upon oxidation as its initial products. They may react either with metals or reduced by glutathione peroxidases to produce aldehydes, epoxides etc and unreactive fatty acid alchohals. Malondialdehyde (MDA), croton aldehyde, 4-hydroxynonenal (HNE) and acrolein are major lipid peroxidation aldehyde reactive products which damage DNA by forming exocyclic adducts. They are also blocking base pairing regions, toxic or mutagenic to DNA. Adduct levels in human and rodent in leucocytes and tissues have been found highly variable. These levels are mostly affected by nutritional intake of fatty acids and antioxidants, inflammation and chronic infections in which levels of nitric oxide (NO) is increased. Intake of high levels of polyunsaturated fatty acids in diet increases malondialdehyde-, etheno- and propano-derived adduct levels in female human leucocyte DNA. Propane and etheno adducts have been detected in rodents treated with carcinogenic compounds and these adducts have proved to be involved in cancer.

Propano adducts

Propano adduct emerges consequently through the reaction of DNA with crotonaldehyde, 4-hydroxynonenal and acrolein, all produced by lipid peroxidation. Acrolein and Crotonaldehyde are produced by fats burning and smoking. Crotonaldehyde react with water molecules to produce 3-hydroxybutanal, which then reacts with DNA to generate the Schiff base N2-(3-hydroxybut-1-ylidene)dG and many N2-paraldol-dG diastereomers. 4-hydroxynonenal is a chief toxic produce by lipid peroxidation (Esterbauer et al., 1991). This HNE modulates the gene expressions involved in apoptosis and cell cycle control. Two scientists Chung and Pan in 2002 proved that the rate of crotonaldehyde adducts is ~5- to 10-times less than that of acrolein adduct formation, depends on the type of polysaturated fatty acids, HNE adducts formation is considerably slower than acrolein adducts. Enals reactivity decreases toward dG as the chain length increases (acrolein > crotonaldehyde > HNE). The propano adducts varies in humans and ranges between 0.0006-0.40 adducts/106 nucleotides.

MDA-induced damage

MDA is a mutagenic and toxic product of lipid peroxidation and prostaglandin biosynthesis. It is highly mutation causing agent in mammalian and bacterial cells and carcer causing agent in rats. M1G or deoxyguanosine adduct has been characterized in white blood cells, liver, pancreas, breast, colon, and from healthy humans and their levels ranges from 0.062 -0.9 adducts/106  nucleotides. Different products have been yield when MDA reacts with DNA and it has been found that among all the possible products, M1G produce five times greater than the amount of M1A. M1G levels also varies in tissues to tissues and increases as age increases and diet content increases in unsaturated fatty acids.

Reactive carbonyl species:

Reactive carbonyl species (RCS) are powerful cellular carbonyl stress mediators that originates from different endogenous chemical processes (glycation and lipid peroxidation). Glycation is a main cause of reactive carbonyl species like methylglyoxal and glyoxal. Methylglyoxal is an important biological carbonyl compound which is a reactive aldehyde compound formed endogenously as a glucose metabolism product. It is also present as a component of a variety of beverages such as coffee and food. It readily forms the cyclic adduct (MG-3’-dGMP) in buccal cells of humans. A scientist Vaca with his team measured 0.26 MG-3’-dGMP adducts/106 nucleotides in 1998. Lipid hydroperoxides products decomposition results in reactive aldehyde glyoxal which can also be produced by autoxidation of sugars or decomposition of oxidative deoxyribose, and it also plays a major role in the ageing and diabetes. It also produces DNA adducts, producing active cancer causing adducts for example glyoxalated deoxycytidine. It has been exhibited as a mutation causing agent in mammalian and bacterial cells. Two scientist Dedon and Awada in 2001 reported 2-phosphoglycolaldehydeformation by a process of oxidation of the 3’-deoxyribose reacts with dG and finally produce 1,N2-glyoxal adducts. Rate of Glyoxal-dG adducts production with 2-phosphoglycolaldehyde occurs five times slower than rate of production with with glyoxal. RCS concentrations have been found high in pathological conditions such as in skin with Sun irradiations and diabetes. Delicate dealing of glyoxal on cultured keratinocytes (human) results in distinct DNA strand breaks, while treating with methylglyoxal results in cross-linked DNA.

DNA hydrolysis

The glycosidic bond among deoxyribose in DNA and bases can be altered under definite conditions of base alkylation, N-glycolases action or heating. Breaking glycosidic bond in DNA causes an abasic site. These abasic sites are not only created by  depurination but also due to reactive oxygen speices. Abasic sites are endogenous lesions widely occur in DNA in 10000 damages/human in each cell per day. Two scientists Swenberg and Nakamura in 1999 showed different levels related to abasic site more or less 50 000 to 200 000 per genome in rodent tissues and humans. Rate of purine lose is 20 times greater than lose of pyrimidines and endogenous abasic sites does not varies within tissues but varies between tissues. These affect mostly brain, colon, heart, liver, kidney and lungs. Atamna with team of scientists in 2000 showed that young human leukocyte contains more than 7 times AP than old human leukocytes samples due to reduction in base excision activity. Pathway named as Type II Abasic site endonuclease-/beta-polymerase-dependent plays important role to repair AP sites.These sites are repaired efficiently and rapidly. Escaped AP sites contributes in chromosomal aberrations, transcription errors and mutations. They typically induce base substitution at Thymine residue. Mutations at 5’-cleaved abasic sites can induce frameshift, such as found in sequences of microsatellite when subsequently treated plasmids with hydrogen peroxide. Abasic sites are mutation causing because during replication they integrate adenine on opposite abasic sites with the help of DNA polymerases.

Oxidative DNA damage

ROS are produced constantly as a result of external factors and internal biochemical and metabolic reactions. ROS comprise singlet oxygen, superoxide, hydroxyl radicals and hydrogen peroxide, and when they oxidize DNA it leads to a variety of DNA lesions, including single/double strand breakdown and oxidation of bases. ROS produces a major occurring DNA damage. This Oxidized DNA needs extensive damage repair in many human tissues, especially in cancer cells/tissues. Resistance mechanisms have been evolved within organism to reduce the levels of ROS and its induced damages. High levels of ROS production creates oxidative stress in the natural antioxidant defence pathways that cause DNA damage and inactivate many antioxidant enzymes. A scientist, Epe in 2002 pointed out that any alteration in the endogenous production of cellular antioxidants or ROS or DNA repair efficiency can cause a consequent modulation of the oxidative DNA level alteration, which sequentially alters the mutagenesis rate and finally the cancer occurrence. Different studies provide evidence of reduced cancer risk, especially in the airways and gastrointestinal tract, linked with antioxidants diet and increased levels of antioxidants in different plasma samples. Different data propose that the rate of DNA damage reduces with increase in age due to decrease in metabolism, while the constant levels increases because of fail in repair mechanisms. ROS production also occurs through a wide range of endogenous mechanisms/pathways. In mitochondrial respiration more or less about 1 to 5% of the oxygen goes through single electron transfer, producing the anion radical of superoxide. This molecule exhibits limited reaction activity but it can be changed to H2O2 by an enzyme, called superoxide dismutase.





Chronic infections that bring out an inflammatory response are effective ROS generators. Oxygen produced by neutrophils are result of hydrogen peroxide and superoxide radicals, that can consequently react with the help of the Haber-Weiss process to produce the effective OH radical. Shen with team in 2000 exposed DNA neutrophils activation or eosinophil activation, which resulted in the production of 8-oxo-dG via a pathway which was blocked enzymes and antioxidant agents. Similarly, peroxynitric  is the nitric oxide product and a neurotransmitter and vascular relaxant, and superoxide. It is generated in small quantities of is in protonated type (peroxynitrous acid) is a tremendously reactive oxidant able to oxidize DNA independent of its capacity to generate HO.. Association between the amount of transversions (G to T) in p53 human colorectal cancers gene and expression levels of nitric oxide synthase enzyme in cancerous and gastric precancerous damages has been studied and found a relationship in disease and high levels of oxidized bases. These diseases include Helicobacter pylori infection, cirrhosis and hepatitis.  




Along with bases modifies via oxidation, 8-oxo-dG is actually the most frequent. Constant levels of 8-oxo-dG have been determined by a wide range of techniques in human cells (ranges between 0.07 to 145.25adducts/106 nucleotides). After replication, it rather than cytosine base pairing, base pairs with adenine to form transversion mutations (GC to TA). Variations in DNA repair potential affects the oxidative DNA damage, and human diseases determined by unusual DNA repair (Bloom's syndrome, Fanconi's anaemia, xeroderma pigmentosum and ataxia telangiectasia) can also be determined through an high levels of 8-oxo-dG. Nitric oxide mediates inflammation, inhibits an important BER enzyme called as hOgg1. This enzyme is responsible for 8-oxo-G BER. This DNA repair inhibition contributes in mutation causing situation for chronic inflammation. Other abundant oxidatively modified bases are the formamidopyrimidine adducts of adenine and guanine: 4,6-diamino-5-formamidopyrimidine (FapyAdenine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGuanine). The latter is especially formed in the absence of oxygen by ionizing radiation and other ROS- producing agents. This lesion is efficiently repaired by a special formamidopyrimidine glycosylase but it can lead to GC toCG transversions. Other modified adenine bases include 8-oxo-adenine (7,8-dihydro-8-oxoadenine) and 2-hydroxy-2’-deoxyadenosine. Oxidation of 2’-deoxycytidine can lead to the formation of 5-hydroxy-2’-deoxycytosine, 5-hydroxy-2’-deoxyuridine and 5,6-dihydroxy-5,6-dihydro-2’-deoxyuridine. Other oxidized bases include 5-hydroxymethyluracil, uracil glycol (formed by oxidative deoxycytosine deamination), thymine glycol and cytosine glycol. Radicals of oxygen react with 5-methylcytosine and oxidize 5,6-double bond.





 Deamination of 5-methylcytosine glycol forms thymine glycol which base pairs with adenine base and results in transition (C toT). It not only a weak mutation causing agent but also it blocks replication and transcription. This damage is mainly repaired by a pathway called BER. Derived from pyrimidine damages, 5-OH-Cyt and 5-OH-Ura had showed a potentially pre-mutagenic damages resulting in transitions (GC to AT) and transversions (GC to CG) (61). 5-OH-Cyt is a most mutation causing oxidative product. 5-Hydroxymethyluracil is formed by oxidizing thymine methyl group. This methyl group is important in a variety of DNA and protein interactions. This molecule then interferes the binding of different transcription factors with DNA. It is a non-mutation causing damage mostly removed by higher organisms which can trigger cell death as a result of break in chromosome and it can cause deletion mutations.

 

Alkylating Agents

Besides oxygen, several other reactive molecules can cause DNA lesions. Among them S adenosylmethionine (SAM) is an important one which donates reactive methyl group and cause enzymatic DNA methylation. It exhibits gene expression regulation function. However, mutation causing adducts can be produced.  Other methylation agents are choline, betaine and simple alkylating agents formed from cellular precursors. DNA methylation produce 3- methyladenine and 7-methylguanine is harmless, due to alteration does not change the base coding specificity. Glycosyl bond destabilization because of substitution N-7 guanine generates muatagenic (AP) sites. Opening of Imidazole ring of 7-methylguanine causes DNA replication ending while 3-Methyladenine is a cancer causing, forms DNA damage which blocks replication. 3-Methylthymine, an alkylating agent can blocks DNA replication and 3-methylcytosine inhibits DNA synthesis and cause mutations. During DNA replication some alkylating agents are highly mutation causing DNA damages of endogenous origin are O4-ethylthymine, O4-methylthymine and O6-methylguanine, they cause transitions (TA to CG and GC to AT). Nitrosated bile salts mainly form N7-carboxymethylguanine upon reaction with DNA. N3-O6-carboxy-methylguanine and Carboxymethyladenine are formed as small products (O'Driscoll et al., 1999). In vivo many methylated adducts can be removed from DNA through BER pathway, which the recognizes altered DNA bases by DNA glycosylases and leaves a AP site.  Abasic sites cause mutations if not repaired. Atamna with team in 2000 showed that old human leucocytes have a reduced glycosylase activity which removes methylated bases. It suggested a decline in BER activity. In 2000, Hemminki and Zhao showed that at constant DNA alkylation levels, alkylating products are age independent and suggested that the endogenous DNA damage ratio, by lipid peroxidation or ethylation, and such damage repair may not be changed in lymphocytes of aged humans