N-Acetylcysteine Promotes DNA Repair after Genotoxic Damage Induced by Copper Sulphate in Human Lymphocytes after Induced

Repair shown that the excess the reactive species. stress also diminishes the DNA repair enzymes, activity, risk. There are existing reports about the role of N-acetylcysteine in DNA protection related to its antioxidant activity; however, its role in DNA repair has not been studied. In the present work, the DNA repair by N-acetylcysteine was analyzed through the comet assay in human lymphocytes after inducing genotoxicity by means of copper sulphate. The gathered data indicates that N- acetylcysteine is innocuous at 1000 µM and significantly reduced the DNA damage in human lymphocytes, which were previously treated with Cu 2 SO 4 , while N-acetylcysteine exerted an adjuvant effect on the repair of DNA in human lymphocytes.


Introduction
The excess of many chemical substances in humans, such as copper has been reported to induce DNA damage and trigger cancer development [1]. The main reason for copper involvement in DNA damage has been associated to the increase of reactive oxygen species (ROS) [2]. ROS can induce DNA damage by means of two mechanisms: generation of DNA mutations, usually base pair substitutions, and DNA cleavage, where single strands breaks induce DNA instability [3]. The comet assay enables detection of the DNA double-strand breaks (DSBs) and it has been used to evaluate copper genotoxicity caused by oxidative damage in several plants, animals [4,5] and human cells as lymphocytes [6,7]. The DNA damage induced by ROS, can be decreased by the antioxidant action of N-acetylcysteine (NAC). Previous studies have reported the effect of NAC on DNA protection from ROS damage [8]. Xie, et al. evidenced the DNA protection effect of NAC against radiation harm on mice lymphocytes [9]. On the other hand, Ansari, et al. reported that NAC protects DNA from oxidative damage in rat intestine [10].
Additionally, NAC has been well established, such as a cytoprotective drug with an antioxidant effect as well as antiinflammatory functions [11]. Therefore, NAC exerts a fundamental roll in DNA protection, which may act as a chemioprotective agent to some diseases [12]. However, if DNA damage already exists, it needs to be repaired because the cell repair system is diminished due to oxidative lesions and this may increase the risk of cancer [13]. Due to the fact that DNA damage can lead towards the initiation of cancer progression, DNA damage repair proteins are present in cells in order to maintain the genome integrity. DNA damage is repaired in two manners: first, lesions are recognized for specialized proteins and second, multiple enzymes are involved in the repair process [14]. Certain studies associate oxidative stress condition with a restricted activity or inactivated enzymes involved in DNA damage repair [15]. Oxidative stress has been reported as the most frequent cause of DNA damage, such being the reason for the NAC to have a protective effect; yet could it be possible that NAC excerts a repairing effect on DNA? The comet assay procedure was followed in order to study the DNA repair effect of the NAC, it was used blood (5ml) from a healthy subject for four times to obtain lymphocytes, afterwards the cells were treated with copper sulphate and NAC.

Objectives
In the present work, we performed the following objectives: 1) To determine the DNA damage caused by copper sulphate.
2) To determine the N-acetylcysteine innocuity/damage to DNA.
3) To evaluate the effect of N-acetylcysteine on DNA repair.

Peripheral Blood Extraction
Peripheral blood was obtained four times from the same healthy subject by venipuncture (5 ml) in a blood sample tube containing EDTA as anticoagulant and it was diluted 1:1 with PBS 1X. We isolated the lymphocytes from the same subject to test the effect of Cu 2 SO 4 and NAC. This procedure was done according to the Mexican bioethics NOM-012-55A3-2012 and following the Helsinki declarations protocol.

Lymphocyte Isolation
Two parts of blood-PBS (1:1) mixture were added to three parts of Ficoll paquepremium. The tube was centrifuged for 30 min at 3500 rpm, and then the white coat was separated and resuspended in PBS at 5 ml final volume. The lymphocytes were centrifuged for 5 min at 2500 rpm and the supernatant was removed. The pellet was resuspended into 1 mL RPMI-1640 medium containing 10% of FBS.

Test Material Treatment
Immediately, after the lymphocyte's isolation, 2 x 10 4 cells per 100 µL of PBS were used to test the effect of Cu 2 SO 4 and NAC. Each tested material was added to the lymphocyte's samples (Table 1) and were incubated at 37 °C for 2 hours. After the treatment, the cells were centrifuged for 5 minutes at 3000 rpm, afterwards, the lymphocytes pellet was resuspended in 100 µL of PBS accordingly, and 10 µL were removed for a trypan blue viability test, additionally, a comet assay was conducted on the samples.

Comet Assay Alkaline Version
The agarose microgels were prepared as follows: the first layer After the electrophoresis time, the microgels were neutralized by placing them twice in a Petri dish with Tris-base (pH 7.0) 5 mL for 2 min. The microgels dried at room temperature and stained with 50 mL of 25 mM ethidium bromide [16]. The genotoxic damage was evaluated by observing the microgels on a fluorescence microscope.
All the experiments were tested in triplicate.

Statistical Analysis
Significant differences between groups were evaluated by twoway ANOVA and a tukey pos hoc test [17].

Evaluation of N-Acetylcysteine Innocuity
In order to evaluate NAC innocuity, three different NAC doses in human lymphocytes were tested (Figure 1). The results did not indicate significant difference compared against the negative control (PBS 1X) at 500 µM and 1000 µM. Even though NAC at 1500 µM had significant differences in regard to the negative control, such dose did not generate the same DNA damage as the positive control (H 2 O 2 ) (p<0.05). It was decided to use 1000 µM for the experiments that followed.

N-Acetylcysteine Effect on DNA Repair
In order to study the effect on DNA repair, N-acetylcysteine  Figure 4. Figure   4A demonstrates a negative control; Figure 4B exhibits positive control; Figure 4C shows NAC 1000 µM; Figure 4D indicates Cu 2 SO 4 200 µM genotoxicity; and Figure 4E proved the NAC effect on the DNA repair.    [21].
Our results are consistent with previous reports about copper genotoxicity in marine animals [22] and mice [23] [8]. A possible explanation for DNA repair by NAC could be the restoration of redox balance due to its antioxidant effect as indicated by previous reports [11,24]. In this case, NAC regulates the redox balance, for the correct function of the enzymatic defense mechanisms, which contains superoxide dismutase, catalase and glutathione-peroxidase as the most important antioxidant enzymes [25].
At the other extreme, antioxidant protection can be nonenzymatic [26]. Glutathione is a non-enzymatic antioxidant composed of L-glutamate, L-cysteine and L-glycine, and is the predominant intracellular non-protein sulfhydryl in a wide range of cells [27]. NAC can promote Glutathione synthesis, and both play a critical role in cellular defense and helps maintain structural and functional viability of proteins, same as DNA repair enzymes, by means of reducing the amounts of ROS by their reducing property through its thiol-disulfide exchange activity [28]. Furthermore, the reduction of ROS leads to the decrease of oxidative DNA damage and enables the enzyme to mediate the DNA repair [29]. It is possible to determine the mechanism by which NAC exerts its action as DNA repair compound by measuring ROS levels and enzymatic activity on DNA repair after treatment with CuSO 4 /NAC.

Conclusion
The conclusion of our collected data indicates that NAC is innocuous to 500 µM and 1000 µM, and CuSO 4 induces DNA damage in a concentration dependent manner in human lymphocytes. The DNA damage caused by CuSO 4 can be repaired by NAC. Based upon these findings, NAC should be evaluated for its innocuity in further studies as well as a source of Geno protective and auxiliary compounds in DNA repair in several other types of cells.

Recommendations
We recommend testing a large range of NAC and CuSO 4 concentrations in different cancer and normal cell lines. However, the measure of ROS levels and the evaluation of enzymatic activity of the DNA repair system could provide additional evidence about the NAC and CuSO 4 Geno protective and genotoxic mechanisms, respectively.