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DNA Adducts

Overview
DNA adducts are reactive compounds that bind onto DNA. Often times, they can interfere with the DNA replication process and contribute to mutagenesis as well as carcinogenesis. They can be distributed anywhere along the genome. Accumulation of DNA adducts are often times associated with higher risks of developing cancer. DNA adducts are “considerably different from mutated or methylated DNA and can be identified in clinical samples with high accuracy and reliability (Grigoryeva et al. 2015, and Phillips et al. 1996).

History
One of the earliest DNA adduct experiments conducted when Kurt Randerath developed an assay that could characterize covalent adducts attached onto DNA. The assay, known as the 32P­labeling test, was used to detect carcinogens and mutagens that were linked to DNA (Randerath et al. 1981). This prompted the start of identifying carcinogenic chemicals within the human DNA.

By the 1990s, 32P­labeling allowed scientists to detect DNA adducts in frequencies of as low as one adduct per 10^10 normal nucleotides (Phillips et al. 2000). The field of analyzing DNA adducts started to emerge as research labs were trying to find correlation between DNA adducts and its association with various types of cancer. At that time, smoking was a huge health issue and the exploration of DNA adducts amongst smokers and non­smokers were being compared. It was discovered that more than 20 different compounds in cigarette smoke contributed to the formation of DNA adducts after undergoing metabolic pathways in human tissue (Jahnke et al. 1990). The field of tracking DNA adducts evidently became more widely known as tracking biomarkers for carcinogenesis.

In the early 2000s, scientists have begun to shift their methods by using mass spectrometry to characterize and quantify DNA adducts. More specifically, the technique is known as Liquid Chromatography­mass spectrometry. Unlike traditional mass spectrometry experiments, this utilized a top­down approach where simultaneous screens for multiple DNA adducts can be derived from known and unknown exposures (Balbo et al. 2014). This is prompting the emergent field of adductomics, where DNA adducts are studied on the genomic scale.

Recently, Manabu Yasui revealed a method to artificially induce DNA adducts into the human genome and track their ultimate fate. The method is tracing DNA adducts in targeted mutagenesis (TATAM) and it utilizes targeting vectors to insert adducts [Yasui et al. 2014]. In their experiment, they used human lymphoblastoid TSCER122 cells that lacked the Thymidine Kinase (TK) gene. A (TK+) vector containing 8­oxog, an oxidative adduct, is inserted into the host cell. After conducting an analysis, it was revealed that 86% of those clone vectors recovered to the regular G nucleobase. The location of the recovery was noted by the increase of MutY activity, which is involved in oxidative DNA damage repair (Parker and Eshleman et al. 2003). The novel approach of TATAM is currently used as a method to identify how DNA adducts get repaired. Sassa and Yasui utilized the method to determine if the nucleotide excision repair pathway is involved in repairing oxidative DNA adduct damage in clustered DNA sites. (Sassa et al 2015). Clustered DNA sites defined as “DNA damage within one or two helical turns of the duplex DNA”. Likewise, they used TSCER122 cells with a XPA knockout group that lacked the NER gene and a control group with XPA. Initially, they measured the two group’s baseline oxoG damage. Afterwards, the vector containing oxoG adducts were inserted to both groups. The final results showed that there was an increase in oxoG mutations in the XPS knockout group, confirming the fact that the nucleotide excision repair is involved in getting rid of clustered DNA damage (Sassa et al 2015).

Methods for screening DNA Adducts
In 1981, the 32­P labeling assay was developed to identify covalent adducts when DNA reacted with carcinogens and mutagens (Randerath et al. 1981). This assay allowed researchers to screen for DNA adducts formed by more than 100 genotoxicants (Balbo et al. 2014). The first step of the assay is to use enzymes to digest DNA into 3’­deoxynucleoside monophosphates. Next, the adducts are labeled with y­32P by the transfer of y­32P from adenosine [y­32P] triphosphate to generate 3’,5’­[5’­32P] diphosphates using T4 poly­nucleotide kinase (Gupta et al. 1981., Balbo et al 2014]. Thin Layer Chromatography (TLC) is then used to separate the adduct nucleotides from the DNA. Finally, the adduct nucleotides are quantified using scintillation counting (Ruberu et al. 2008).

A major advantage of the 32­P labeling assay is that it could be employed in human studies where subjects are exposed to hazardous chemicals present in the environment. (Balbo et al. 2014). However, this also comes with an advert risk as radioactive exposure to human subjects may cause detrimental health effects. The main disadvantage is that the assay does not provide any structural information for positive identification of unknown adducts (Klaene et al. 2013).

Currently, the main technique to identify DNA adducts is to use liquid chromatography­mass spectrometry. Its main advantage is that DNA adducts can be identified through mass spectrometry even if the adduct was never previously documented [Klaene et al. 2013, Balbo et al. 2014]. Moreover, a newer iteration of the technique is called Ultra performance liquid chromatography mass spectrometry (UPLC). This technique is efficient as analysis can be conducted in less than 10 minutes and there’s a resolution separation efficiency of (>100,600) plates (Klaene et al. 2013]. While there have been recent advancements in characterizing the structures and identifies of DNA adducts, it is still difficult to determine the molecular mechanism in regards to forming DNA damage (Delaney and Essigmann 2008). The greater challenge that scientists face today is to understand all the pathways that the DNA adducts are part of and how to prevent repair all these deleterious components that have the potential to alter the DNA sequence of an organism.

Types of Carcinogenic DNA adducts
Currently, there are for distinguished types of DNA adducts that cause carcinogens. They are N­nitroasmines, aromatic amines, and polycyclic hydrocarbons. (Beland and Poirer 1989). More specifically, there has been extensive research done to figure out the associations between aromatic amines, and polycyclic hydrocarbons with a multitude of different types of cancer.

­Nitroasmines are chemical compounds that are most commonly found cosmetics, foods, and rubber products (Beland et al. 1994). The most deleterious nitroasmines include 4­(methylnitroasmino)­1­(3­pyridyl)­1­buanone (NNK), and N’­nitrosonornicotine (NNN) (Hecht 1999). It is activated by P450­catalyzed oxidation which results in the alkylation of DNA. This ultimately results in the formation of a DNA adduct and errors forming within the DNA replication step (Peterson et al. 2014). Next, one of the most common and prevalent DNA adduct is aromatic amines more commonly known as bulky DNA adducts.. Individuals may be exposed to aromatic amines through cigarette smoke and industrial processes (Beland and Kadlubar, 1990). These bulky DNA adducts are correlated with having higher chances to develop carcinogenesis (Veglia et al. 2003). On a granular scale, a meta­analysis was conducted to determine whether or not smokers have higher levels of DNA adduct on the individual scale. Likewise, the Veglia and her team concluded that individuals who were currently smoking in the studies had 83% higher levels of adducts than the nonsmoker group (Veglia et al. 2003). Moreover, this was correlated with an increased risk of developing lung cancer because benzo[a]pyrene adducts were found to at mutation hotspots for the p53 gene (Denissenko et al. 1996). The p53 is a tumor suppressor gene that plays a vital role to prevent cells from replication if they accumulate too much DNA damage. The gene regulates whether or not a cell would go through apoptosis or continue on with cellular replication (Ji et al 2015). Moreover, the mutation of p53 is commonly as a biomarker for the start of carcinogenesis. In Denissenko’s (et al 1998) paper, the researchers saw that the DNA repair of on the transcribed DNA strand occurs quicker than the non­transcribed DNA strand. Moreover, they also saw that the DNA adduct accumulated greatly within the hotspots;benzo(a)pyrene diol expoxide (BDPE) is the main DNA adduct that was tested for in the experiment. A recent study in 2015 looked at the mutation patterns of TP53 in lung cancer and found that BDPE adducts were most associated with codon 157 which is a hotspot (Menizies et al. 2015)) Overall, this highlights how bulky DNA have a major impact in the start of carcinogenesis.

Another prominent bulky DNA adduct is the polycyclic aromatic hydrocarbons (PaHs). Humans are often exposed to PaHs through cigarette smokes. PaHs are known to be biomarkers for lung cancer (Talaska et al. 1996). Individuals who work within the industrial sector are seen to have higher prevalence for developing lung cancer. Interestingly enough, the genotoxicity level is based on the structural component of PaHs. PaHs that contain higher amounts of benzene rings and greater structural complexity are more susceptible to binding with DNA (Bostrom et al. 2002). In other words, PaH’s abilities to bind with DNA determines its genotoxicity levels. The human body’s system to remove PaH’s consists of utilizing an enzyme known as apyrimidinic endonuclease 1 (APE1). A 2015 study conducted on Chinese male workers showed that humans with the APEX 148Glu, a variation, were more susceptible to lung cancer (Li et al. 2015). This study gave consistency that the APEX gene is indeed involved in the repair process for PaHs.

Overall, various studies have been conducted to correlate the appearances of DNA adducts within human tissues to lung cancer. It is widely known that roughly 20 substances in cigarette smoke contribute to a cariogenic effect. Studies have revealed that specific adducts related to increased levels of adducts in the lung. They include, but are not limited to polycyclic aromatic hydrocarbons and nitrosamines. These compounds are involved with metabolic pathways that leads to genotoxic actions that may be interpreted as “a cumulative mirror of heterogeneous response of different individuals to smoking carcinogens”. Grigoryeva et al. 2015). Future directions for studying DNA adducts in lung cancer include exploring the possible adduct threshold levels before individuals are characterized with cancer.

Future Directions of Adductomics
The first scientific journal utilizing the term “adductomics” was around 2010. This new and emerging field is focused on studying DNA adducts on the genomic level (Spilsberg et al 2010). The first group of scientists that leveraged adductomics conducted a study to detect DNA adducts that potentially arises from foods. It is widely known that damaged nucleosides arise over the span of a human lifetime due to various such as diseases, aging, and environmental influences. The study focused on analyzing damaged nucleosides that arrived from the consumption of quorn. The results from the adductomic approach showed that foods gave rise to many different DNA adducts. (Spilsberg et al 2010).