**2. Basic biochemistry of ICL-generating agents**

*DNA - Damages and Repair Mechanisms*

mono-adducts, intrastrand cross links as well as DNA-protein cross-links [3]. The ICLs are made with the help of reactive endogenous chemicals such as lipid peroxidation product known as malondialdehyde or aided with the reactive aldehyde group of an unpromptedly formed or the enzyme-derived abasic site in the DNA

A large amount of anticancer and chemotherapeutic agents such as mitomycin C (MMC), cisplatin, nitrosoureas and nitrogen mustards are notorious for introducing formidable blocks in the normal metabolic processes of DNA with ICLs and need repair for cell sustenance. ICLs are also caused by various antitumor agents that defects DNA through radical processes like C-1027, neocarzinostatin [5]. With the passage of time, the organisms have developed various complex mechanisms to alleviate these deleterious defects from the genome. The failure to remediate the defect can contribute towards cell death that can occur either through a mitotic catastrophe or the p53-dependent apoptotic pathway. In the mammalian cells, the repair mechanisms for ICLs repair are still ambiguous [6]. According to an estimation, about 40ICls that form in a mammalian genome can destroy a defective cell that lacks ability to be repaired. The in vivo study gives an overview of the elimination of the ICLs in cellular DNA of both prokaryotes and eukaryotes. The model organisms are used for the clear understanding of the repair mechanisms. These include *E. coli* and yeast. The ICLs repair mechanisms in bacteria and yeast are replication dependent and independent while in vertebrates, they follow repairment pathway during replication of DNA [7]. Moreover, the recent study suggests the operation of replication

The ICL repair pathway have been deduced from the relative sensitivity of the DNA repair defective cell lines to the cross linking agents. Pathways of ICL repair have mostly been inferred from the sensitivities of DNA repair defective cell lines to crosslinking agents. During the S phase of the cell division in vertebrates, the ICL repair is induced by the help of impeded replication forks. The process of ICL repair needs a nexus of multiple factors along with the structure specific endonucleases, for example TLS and HR. If a disturbance occurs during the repair, the genomic instability results that bring forth the birth of Fanconi anemia, a cancer prone ailment [8]. There is another ICL repair pathway that takes place in the G0/1 phase during the cell cycle which is a replication and recombination independent pathway [9]. In addition, the tolerance of ICLs in G1 as compared to S phase makes it an underappreciated pathway because there, the stalled replication fork possesses high toxicity. Contrarily, the toxicity of ICL in G1 can be depicted when it terminates the

The latest studies have proposed the role of NER proteins (as they cut one side of ICL) [7], Homologous recombination along translesion synthesis polymerases (Polζ, Rev1) that are involved in filling the gap for both type of cells undergoing replication as well as non-replicating ones [10]. The proteins involve in the ICL repair have a vital role in the pathophysiology of several hereditary diseases Proteins implicated in the repair of ICLs have a critical role in the pathophysiology of several hereditary disorders. In addition, cells deficient in the Fanconi Anemia (FA) pathway are highly sensitive to ICLs [11] and this pathway has been suggested to play an important role in mammalian ICL repair at replication forks promoting homologous recombination. There has been a series of continuous research on ICL lesions in the past decade and it covered the various aspects of ICLs be it as their identification, detection methods or their development along with the repair mechanisms and the exploitation of cross linkers in the laboratory. These have paved the way towards the better and more reliable understanding of ICLs in the complex biological samples. This chapter foregrounds the multiple aspects of the interstrand cross-link repairs

with a reference to their pathophysiology and lesion repair mechanisms.

molecule with a normal base on the complementary strand [4].

independent ICL repair pathway in vertebrates.

transcription of a gene playing a vital role.

**122**

A large variety of natural and synthetic chemicals are notorious for bringing ICLs on the front and are regarded as the ICL inducers or inducing agents. In the same way, the metabolic byproducts formed in the cell also contribute towards ICLs formation. Their structure and function vary greatly but ICLs inducers are known for their bifunctional reactivity with both of the strands of DNA. The endogenous as well as exogenous sources of ICLs are summarized as follows:

## **2.1 Endogenous sources of interstrand cross links**

The endogenous sources of ICLs comprises of the reactive aldehydes that are generated as a result of lipid peroxidation along with base excision repair (BER) [12]. There are other endogenous by products of lipid peroxidation, the α, β-unsaturated aldehydes or enals namely crotonaldehyde, acrolein, along with the 4-hydroxynonenal (4-HNE). These are formed as a result of oxidative stress [13]. Moreover, there are exogenous contributors as well namely cigarette smoke and automobile exhaust to expose with acrolein and croton-aldehyde. The DNA nucleobases interact with enals to give rise to exocyclic adducts. These adducts then interact with proteins. The incorporation of enals to dG is done with the help of Michael addition in which addition of N2 -amine occurs to generate N2 - (3-oxopropyl)-dG adducts. The next stage is cyclization of N1 with the aldehyde, giving rise to N2 -γ-hydroxypropano-dG adducts [14]. These products are also genotoxic to human beings. Shapiro and Leonard are famous for their earlier study of nucleosides reactions with glyoxal, chloroacetaldehyde, malondialdehyd along with related bis-electrophiles [14, 15]. The in vitro formation of ICL is attributed to the opening of the exocyclic 1, N2 -dG product that minimizes the steric hindrance and forms ICL on exposure towards an aldehyde [16].

Moreover, there are DNA lesions that are formed as a result of accumulated acetaldehyde in the cells. The acetaldehyde is produced as a result of alcohol metabolism with aldehyde dehydrogenase 2 (ALDH2) as a biocatalyst. The drug disulfiram if used, blocks the enzyme ALDH2 and accumulates the acetaldehyde in the cells. The lesions produced are DNA adducts, breaks in single or double-strands of DNA (DSBs), sister chromatid exchanges (SCEs), point mutations, along with crosslinks in DNA [17]. The DNA adducts like *N*<sup>2</sup> -ethylidene-2′-deoxyguanosine, *N*2 -propano-2′-deoxyguanosine, *N*<sup>2</sup> -ethyl-2′-deoxyguanosine, along with *N*<sup>2</sup> etheno-2′-deoxyguanosine are vital DNA damage agents that follow the accumulation of acetaldehyde in the cells. The acetaldehyde reacts with guanine and forms a crosslink precursor known as N2 -propanoguanine (PdG) which in turn reacts with N2 amine of guanine in 5′-CpG sequence consequently forming acetaldehyde interstrand crosslinks (AA-ICL). In Asian continent, the irreparable detoxification of acetaldehyde is found more often and is linked with alcohol mediated cancers [18]. Moreover, cells in *Saccharomyces cerevisiae* don't have ability to repair ICLs and are acetaldehyde sensitive thus gives validation of acetaldehyde mediated ICLs [19].

The intestinal pathogens in human beings known as *Enterobacteriaceae* and other bacteria play a vital role in the progression of colorectal cancer. They produce colibactins that are genotoxic in nature and bring harm to human beings. With their structural chemistry still unknown, colibactins produce ICL dependent DNA double-strand breaks (DSBs) and activates the ICL repair pathways [20]. *Cellulo* also depicts another picture of the DNA damaging mechanism in which colibactin producing bacterial exposure towards the genomic DNA of cultured human cells made it susceptible to interstrand cross links. There are different changes observed in the intoxicated cells including the replication stress, the activation of


#### *DNA - Damages and Repair Mechanisms*

**125**

*Interstrand Crosslink Repair: New Horizons of DNA Damage Repair*

follow to contribute in various therapeutic applications.

and 531 bending into its major groove [24].

agents of Interstrand crosslink lesions.

**3. ICL Repair genes and human disorders**

total DNA lesions [23].

ataxia-telangiectasia along with Rad3-related kinase (ATR), as well as the retrieval of Fanconi anemia protein D2 (FANCD2). Contrarily, FANCD2 knockdown or ATR inhibition decreases the survival capability of cells having an exposure towards colibactins. The evidence ensures that collectins mediated DNA defects in infected

The other sources of ICLs are exogenous in nature. They have the same mechanism of bifunctional alkylating agents but differ in their preferences for sequences, topologically restrict the DNA and need certain processing within the cell to form functioning ICL inducers [9]. In spite of the fact that they have a history of damaging DNA, their innovative uses also aid in understanding the mechanisms they

These include psoralens that belong to the family of furocoumarins, being mutagenic are still a matter of contention with their photochemotherapeutic applications in inflammatory skin diseases like psoriasis, vitiligo and eczema [22]. The Psoralens generates adducts on interaction with pyrimidines, most often with thymine and give rise to ICLs at the sequences made up of d(TpA):d(TpA) residues [23]. The several derivatives of psoralen form multiple changes in the DNA helical structural framework and exhibit their toxic nature. The DNA duplex adducted with 4′- (aminomethyl)-4,5′,8-trimethylpsoralen (AMT) exhibited 561 unwinding

Another chemotherapeutic agent known as cis-platinum diamminedichloride i-e CDDP, cisplatin also induces ICLs. It makes an adduct with purines, most often at the N7 position of the guanines, hence ICL forms at d(GpC): d(GpC) sequences. This is employed in various head and neck cancers, esophageal, epithelial lung, colon, gastric, bladder along with ovarian and testicular tumors. About 90% of the total defects are formed by 1,2-IaCL and 1,3-IaCL along ICL making only 5% of the

Apart from these anticancer agents, one of prime importance is Adriamycin which is also termed as doxorubicin. It generates a great response against a range of tumors be it as breast tumors, acute leukemia, lymphomas, stomach, sarcomas, multiple myelomas or bone tumors. It is employed as a singly or in combined form [25]. The interaction of Adriamycin is clearly understood with the help of the *in vitro* transcription assays that demonstrates the drug-induced DNA adducts at the GpC sites [26]. The electrospray mass spectral analysis revealed details of GpC drug binding regions and gives the information that the cross links are favored by formaldehyde under the certain conditions [27]**. Table 1** illustrates the exogenous

The proteins involved in the repair of ICLs have vital role in pathophysiology of various hereditary disorders for example xeroderma pigmentosum (XP), cerebrooculo-facio-skeletal syndrome (COFS), Fanconi Anaemia (FA), trichothyodistrophy as well as Cockayne syndrome (CS) [37]. FA is associated with aplastic anemia, cancers (often acute myelogenous leukemia) and bone marrow failure. The mutational changes in any *FANC* genes contribute towards genomic instability and the sensitivity against the ICL agents [38]. According to an estimate 18 genes are involved in FA and the products of genes collaborate for ICL repair during the S phase [39]. Apart from these, the defective NER pathways also result in several rare

*DOI: http://dx.doi.org/10.5772/intechopen.97551*

cells favors DNA ICLs [21].

**2.2 Exogenous sources of ICLs**

**Table 1.** *Exogenous agents of Interstrand cross-link lesions.*

ataxia-telangiectasia along with Rad3-related kinase (ATR), as well as the retrieval of Fanconi anemia protein D2 (FANCD2). Contrarily, FANCD2 knockdown or ATR inhibition decreases the survival capability of cells having an exposure towards colibactins. The evidence ensures that collectins mediated DNA defects in infected cells favors DNA ICLs [21].
