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. Author manuscript; available in PMC: 2018 Apr 1.
Published in final edited form as: Toxicol In Vitro. 2017 Jan 28;40:336–346. doi: 10.1016/j.tiv.2017.01.020

Copper-mediated DNA damage by the neurotransmitter dopamine and L-DOPA: A pro-oxidant mechanism

Nida Rehmani a, Atif Zafar a, Hussain Arif a, Sheikh Mumtaz Hadi a, Altaf A Wani b,c,d,*
PMCID: PMC5404347  NIHMSID: NIHMS853439  PMID: 28137434

Abstract

Oxidative DNA damage has been implicated in the pathogenesis of neurological disorders, cancer and ageing. Owing to the established link between labile copper concentrations and neurological diseases, it is critical to explore the interactions of neurotransmitters and drug supplements with copper. Herein, we investigate the pro-oxidant DNA damage induced by the interaction of L-DOPA and dopamine (DA) with copper. The DNA binding affinity order of the compounds has been determined by in silico molecular docking. Agarose gel electrophoresis reveals that L-DOPA and DA are able to induce strand scission in plasmid pcDNA3.1 (+/−) in a copper dependent reaction. These metabolites also cause cellular DNA breakage in human lymphocytes by mobilizing endogenous copper, as assessed by comet assay. Further, L-DOPA and DA-mediated DNA breaks were detected by the appearance of post-DNA damage sensitive marker γH2AX in cancer cell lines accumulating high copper. Immunofluorescence demonstrated the co-localization of downstream repair factor 53BP1 at the damaged induced γH2AX foci in cancer cells. The present study corroborates and provides a mechanism to the hypothesis that suggests metal-mediated oxidation of catecholamines contributes to the pathogenesis of neurodegenerative diseases.

Keywords: Copper, L-DOPA, Dopamine, γH2AX, DNA damage, Neurotoxicity

Graphical abstract

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1. Introduction

The genome of living organisms is continually exposed to physical and chemical DNA damaging agents, of both endogenous and exogenous origins. Although catecholamines have been recognized as potent radical scavengers (Schimizu et al., 2010), evidence suggests that these metabolites can cause significant oxidative damage under certain conditions (Ando et al., 2011; Tanaka et al., 1991). Further, compelling studies have provided support for the autotoxicity, i.e. inherent cytotoxicity of catecholamines and related metabolites (Goldstein et al., 2014). In recent years, several in vitro and in vivo studies have documented the toxicity of dopamine (DA) and L-DOPA.

Dopamine, a chemical neurotransmitter accounts for about 90% of the total catecholamines in the central nervous system (CNS) (Husain and Hadi, 1995). According to a recently popular hypothesis, the continued death of dopaminergic neurons in Parkinson’s disease (PD) may be the consequence of aberrant oxidation of dopamine via production of DNA reactive species (Snyder and Friedman, 1998). Accumulating evidence suggests that DA and related metabolites are capable of causing neuronal toxicity (Cheng et al., 1996) via generation of quinone derivatives (Graham et al., 1978) and free radicals (Rossor et al., 1980). Further, dopamine is the most biologically abundant catecholamine, with concentrations in the human and mouse brain around 8 μM. (Musshoff et al., 2005; Naka et al., 2002). L-DOPA, the precursor metabolite for the neurotransmitter DA is considered the most effective therapeutic agent for PD (Llyod et al., 1975). It has been reported that the brain concentrations of L-DOPA can be measured to 12 μM in rats after chronic administration (Chalmers et al., 1971). However, research has revealed the long-term complications caused by L-DOPA in the progression of PD while some studies have implied that L-DOPA increases life expectancy (Pardo et al., 1995). Some reports have implied that L-DOPA may accelerate the deterioration of Parkinsonian patients, and that L-DOPA toxicity occurs in damaged dopaminergic neurons in vivo (Ogawa et al., 1994; Blunt et al., 1993; Walkinshaw et al., 1995). It is noteworthy that L-DOPA production from tyrosine is mediated by a copper containing enzyme, tyrosine hydroxylase, presenting the possibility of copper chelation (Perveen et al., 2014).

Multitude of reports have indicated the elevated concentration of labile metals in neurological disorders, generating interest to elucidate metal-neurotransmitter interactions (García et al., 2012). Halliwell and colleagues had suggested that metal ion release could be an important mechanism for the neurotoxicity of L-DOPA and related metabolites (Spencer et al., 1994). Further, transition metal ions have been reported to promote the toxicity of catechols, including L-DOPA (Halliwell, 2009, 1989). It has also been ascertained that the labile concentrations of copper are elevated in various malignancies (García et al., 2012). Evidence has indicated the increased redox auto oxidation of catechols in the presence of copper (Jung and Surh, 2001).

Copper is known to possess the highest redox activity among the transitions metals, facilitating the production of reactive oxygen species (ROS) (Hadi et al., 2007). Moreover, the tissue, cellular and serum copper levels are known to be significantly elevated in various malignancies (Ebadi and Swanson, 1987; Yoshida et al., 1993). It is also well established that copper is prevalent in brain, especially, in basal ganglia, hippocampus, cerebellum, synaptic membranes and cerebellar granular neurons (Desai and Kaler, 2008; Madsen and Gitlin, 2007). The CNS is extensively rich in enzymes, which depend on copper for their functioning, e.g., tyrosinase, copper/zinc superoxide dismutase, ceruloplasmin and cytochrome c oxidase (Rinaldi, 2000). Evidence indicates that there is a relation between copper, its abnormal homeostasis and the pathogenesis of several neurological diseases. Since approximately 20% of total copper is stored in nucleus (Agarwal et al., 1989), DNA is highly susceptible to copper catalyzed reactions. The studies by Pall et al. indicated that copper dependent oxidative mechanisms might contribute towards the pathogenesis of PD (Pall et al., 1987). It is noteworthy that cancer cells tend to accumulate high levels of copper, which is presumed to be crucial for metastasis and angiogenesis (Gupte and Mumper, 2009). Interestingly, colon cancer HCT116 cells (used in this study) accumulate 7-fold more copper than normal cells (Fatfat et al., 2014).

Over the years, we have demonstrated that several of the biological antioxidants, e.g. L-DOPA, uric acid, bilirubin, tannic acid and flavonoids (Asad et al., 1999; Bhat and Hadi, 1994; Fazal et al., 1990; Shamsi and Hadi, 1995) are capable of acting as pro-oxidants. Towards the goal of understanding the chemical basis for the cytotoxicity of endogenous metabolites, this study examines and compares the DNA reactive activities of the neurotransmitter DA and its metabolic precursor, L-DOPA in the presence of copper. Our results suggest a putative mechanism for the toxicity of catecholamines in light of physiologically elevated copper. Moreover, this report highlights the relevance of understanding endogenous genotoxic mechanisms in developing treatment strategies for oxidative DNA damage mediated disease conditions.

2. Materials and methods

2.1. Chemicals

L-DOPA, DA, calf thymus DNA, agarose, low melting point agarose (LMPA), EDTA, bathocuproine, neocuproine, Histopaque 1077, RPMI 1640, phosphate buffered saline (PBS) Ca2+ and Mg2+ free, Triton X-100 and Trypan blue were purchased from Sigma (St. Louis, USA). All other chemicals used were of analytical grade. The plasmid pcDNA3.1 (+/−) was isolated using the GenElute HP plasmid maxiprep kit from Sigma. L-DOPA and DA were dissolved in double distilled water as 1–2 mM stock solutions prior to experimentation. Upon addition to reaction mixtures, in the presence of buffers and at the concentrations used, the compounds remained in solution. Further, the volumes of stock solution added did not result in any appreciable change in the pH of reaction mixtures.

2.2. Fluorescence studies

The fluorescence studies were performed on a Shimadzu spectrofluorophotometer RF-5310 PC (Kyoto, Japan) equipped with a plotter and a calculator. The compounds (L-DOPA and DA) were excited at the wavelengths indicated in the legends and emission spectra were recorded in the range as shown in figures.

2.3. Reduction of Cu(II) to Cu(I) by L-DOPA/DA

The selective copper sequestering agent, neocuproine, was employed to detect the reduction of Cu(II) to Cu(I) spectrophotometrically by recording the spectra between 300–550 nm. The reaction mixture (3.0 ml) contained 10 mM Tris–HCl (pH 7.5), 50 μM L-DOPA/DA, 100 μM Cu(II), and 300 μM neocuproine. The reactions were started by the addition of Cu(II) and the spectra were recorded immediately afterwards.

2.4. Molecular Docking studies

Docking studies between compounds (L-DOPA, DA) and double strand DNA having a sequence of d(CGCGAATTCGCG)2 dodecamer (PDBID: 1BNA) were done with the standard AutoDock (v4.2) suit using Lamarckian Genetic Algorithm (Morris et al., 2009; Peng et al., 1996). The target B-DNA molecule and individual ligands were prepared using standard docking protocol and were saved as ‘PDBQT’ format. In docking calculations, the target-ligand conformations generated were ordered using an energy based scoring function. To identify the potential binding sites of individual ligands in DNA as target, blind docking was performed. The input ‘grid parameter’ files were modified and the grid size was amended to X = 56, Y = 66 and Z = 110 with 0.375Å grid spacing. All the other docking parameters were set to default values. After docking, the top pose conformation of each docked ligand was visualized with the help of PyMOL software (Molecular Graphics System, version 1.5.0.1, Schrodinger.LLC), to determine the possible interactions between ligand and DNA (DeLano, 2002).

2.5. Treatment of supercoiled plasmid pcDNA3.1 (+/−) with compounds

Reaction mixtures (30 μl) contained 20 mM Tris-HC1 (pH 7.0), 200 ng of plasmid pcDNA3.1 (+/−) and other components as mentioned in the legends. Incubation at room temperature was performed for 1 hr at 37°C. After incubation, 10 μl of a solution containing 40 mM EDTA, 0.05% Bromophenol blue tracking dye and 50% (v/v) glycerol was added and the solution was subjected to electrophoresis on 1% agarose gels. The gels were stained with ethidium bromide (0.5 μg/ml), viewed and photographed on a UV light transilluminator.

2.6. Isolation of viable lymphocytes

Fresh heparinized blood samples (2 ml) from a nonsmoking healthy donor were obtained by venepuncture and diluted suitably in Ca2+ and Mg2+ free PBS. Lymphocytes were isolated from blood using Histopaque 1077 (Sigma) and the cells were then suspended in RPMI 1640. The lymphocytes were assessed for their viability before the start and after the end of the reaction using Trypan Blue Exclusion Test (Pool-Zobel et al., 1993). The viability of the cells used in the experiments was found to be greater than 93%.

2.7. Lymphocyte treatment and estimation of DNA breakage by alkaline single-cell gel electrophoresis (comet assay)

Lymphocytes isolated from 2.0 ml blood sample were diluted to the count of 2 × 105 cells/2 ml using RPMI 1640. Approximately, 10,000 cells were then mixed with 75 μl of 1% low melting point agarose (pre-warmed) in PBS. This mixture was immediately applied to a frosted microscopic slide layered with 75 μl of 1% standard agarose in PBS. Subsequently, the slides were covered with coverslips and kept on ice for 10 min to solidify the agarose. Lymphocytes were exposed to different concentrations of compounds in the absence and presence of copper specific chelator, neocuproine 50 μM in a total reaction volume of 1 ml (400 μl RPMI, Ca2+ and Mg2+ free-PBS, and indicated concentrations of compounds) and processed further for comet assay. Comet assay was performed under alkaline conditions according to the procedure of Singh et al. (1988) with slight modifications as described in our previous studies (Khan et al., 2011). Slides were scored using an image analysis system (Komet 5.5, Kinetic Imaging, Liverpool, UK) attached to an Olympus (CX41) fluorescent microscope and a COHU 4910 (equipped with a 510–560 nm excitation and 590 nm barrier filters) integrated CC camera. Comets were scored at 100× magnification. Images from 50 cells (25 from each replicate slide) were analyzed. The parameter used to assess lymphocytes DNA damage was tail length (migration of DNA from the nucleus, μm), which was automatically generated by Komet 5.5 image analysis system.

2.8. Cell culture and reagents

Human glioblastoma cells Gli36, human colon carcinoma cells HCT116 and normal skin fibroblasts OSU2 were maintained according to established methods as described. Gli36 cell line was grown in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (50 units/ml penicillin and 50 μg/ml streptomycin). HCT116 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS and antibiotics as above. Normal human skin OSU-2 fibroblasts were established and maintained in culture as described in previous studies (Venkatachalam et al., 1995). All cells were grown at 37°C in a humidified 5% CO2 atmosphere. The DC protein quantitation reagents were from Bio-Rad. Antibodies against the following proteins were diluted in blocking buffer: mouse β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1/3000; rabbit phospho-histone H2A.X, serine 139 (Millipore, Billerca, MA) diluted 1/3000. Alexa Fluor 680-conjugated anti-rabbit IgG (Molecular Probes) and IRDye800 conjugated anti-mouse IgG (Rockland, Gilbertsville, PA) were used as secondary antibodies. Texas red and FITC conjugated fluorescent antibodies were from Santa Cruz Biotechnology.

2.9. Western blot analysis

Cells were treated with L-DOPA/DA for 1 hr in serum free medium. Following treatment, cells were harvested by brief trypsinization and pelleted by centrifugation. The pellet was washed with PBS and centrifuged again. Depending on the size of the pellet, 75–150 μl of SDS lysis buffer (50 mM Tris HCl (pH 6.8), 2% SDS, 10% glycerol) was added. The lysed cells were then boiled for 5 minutes, cooled to room temperature and centrifuged once again. The supernatant was removed and transferred to a 1.5 ml centrifuged tube. The lysate (supernatant) was quantified by protein assay (Dc Protein Assay System; Bio-Rad, Hercules CA). After protein quantification, equal amounts of protein samples (25 μg) were loaded and separated on a polyacrylamide gel (15% w/v). Proteins were transferred to nitrocellulose membranes via electrophoresis and blocked with 5% non-fat dry milk in Tris-buffered saline Tween (M-TBST; 20 mM Tris, 0.5 M NaCl, and 0.05% Tween 20 [pH 7.4]) for 1 hr at 37°C. Membranes were incubated overnight at 4°C with a primary antibody, followed by washing three times with TBST buffer then incubated with the appropriate secondary antibody at 37°C for 1 hr. The membranes were washed with TBST buffer and analyzed by the Odyssey infrared image system (LiCor). β-Actin on the same membrane, and processed in parallel, was used as a loading control.

2.10. Immunofluorescence Microscopy

The immunofluorescence staining was performed according to established methods (El-Mahdy et al., 2006). In brief, cells on coverslips were subjected to different treatments as demonstrated in Fig. 12, for 1 hour. The cells were then washed twice with cold PBS, permeabilized with 0.5% Triton X-100/PBS for 8 min on ice and fixed with 2% paraformaldehyde in 0.5% Triton X-100 at 4°C for 3 min. The fixed cells were rinsed twice with cold PBS and blocked with 20% normal goat serum (NGS) in 0.1% TritonX-100/PBS buffer at 4°C, overnight. The coverslips were stained with the following antibodies prepared in washing buffer containing 5% NGS for 1 hr at room temperature-primary mouse phospho-histone H2A.X (1/200 dilution), rabbit anti-53BP1 (1/300) and secondary goat anti-mouse Texas Red (1/200), goat anti-rabbit Fluorescein Isothiocynate (1/200) conjugated antibodies. After each antibody incubation step, the cells were washed with 0.1% Tween-20 in PBS 4 times for 10 min each. Fluorescence images were obtained with a Nikon Fluorescence Microscope E80i (Nikon, Tokyo, Japan) and processed with SPOT analysis software.

Fig. 12. Immunofluorescent detection of 53BP1 and γH2AX foci after treatment with compounds, A) L-DOPA and B) Dopamine.

Fig. 12

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Gli36 cells were treated with (1mM) L-DOPA/dopamine in the absence and presence of (200 μM) Cu(II) and processed for immunofluorescence. Phleomycin was used as a positive control for this experiment. Slides were immuostained with γH2AX and 53BP1 antibodies. The merged images show co-localization of γH2AX and 53BP1 at the damage site. Nuclei of cells are illustrated with DAPI staining (blue fluorescence). Calibration bar is 10 μm.

2.11. Statistics

The statistical analysis was performed as described by Tice et al. (2000) and is expressed as mean ±SEM/SD of three independent experiments. A Student’s t-test was used to examine statistically significant differences. Analysis of variance was performed using ANOVA. P-values < 0.05 were considered statistically significant.

3. RESULTS

3.1. Binding of copper ions to L-DOPA/DA

The interaction of copper ions with L-DOPA and DA was investigated by studying the effect of increasing Cu(II) concentrations on the fluorescence emission spectra of these compounds. The results shown in Fig. 1 demonstrate that the addition of Cu(II) to L-DOPA/DA causes quenching in fluorescence emission of these compounds. Further, a decrease in the degree of fluorescence is observed with increasing concentrations of Cu(II). These results clearly indicate that Cu(II) is able to bind with both, L-DOPA (Fig. 1A) and DA (Fig. 1B).

Fig. 1. Effect of increasing copper concentrations on the fluorescence emission spectra of A) L-DOPA and B) Dopamine.

Fig. 1

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L-DOPA and dopamine (in 10 mM Tris-HCl, pH 7.5) were excited at 280 and 278 nm, respectively in the presence of increasing concentration of Cu(II). The emission spectra were recorded between 290 and 450 nm.

3.2. Formation of complexes involving calf thymus DNA with L-DOPA/DA

Fig. 2 shows the effect of increasing DNA molar base pair ratios on the fluorescence emission spectra of L-DOPA and DA, excited at 280 and 278 nm, respectively. Such an addition resulted in a dose dependent quenching of fluorescence of L-DOPA (Fig. 2A) and DA (Fig. 2B). However, no significant shift in the λmax emission implied a simple mode of interaction of DNA with L-DOPA/DA.

Fig. 2. Effect of increasing concentrations of native calf thymus DNA on the fluorescence emission spectra of A) L-DOPA and B) Dopamine.

Fig. 2

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L-DOPA and dopamine (in 10 mM Tris-HCl, pH 7.5) were excited at 280 and 278 nm, respectively in the presence of increasing native DNA base pair molar ratios. The emission spectra was recorded between 290 and 450 nm.

3.3. Detection of L-DOPA/DA-induced Cu(I) production by neocuproine

The formation of Cu(I) produced as a result of reduction of Cu(II) by L-DOPA and dopamine, was analyzed using neocuproine. Neocuproine, a selective Cu(I) sequestering agent, binds specifically to the reduced form of copper, i.e., Cu(I), but not to the oxidized form, Cu(II) (Simpson et al., 1992). The Cu(I)-neocuproine complexes exhibit an absorption maximum at 450 nm. As evident from Fig. 3, the compounds (L-DOPA/DA), or Cu(II), do not lead to any change in spectra with neocuproine. However, L-DOPA+Cu(II) (Fig. 3A) and DA+Cu(II) (Fig. 3B) react to produce Cu(I), which complexes with neocuproine resulting in peaks appearing at 450 nm. The results suggest that these compounds possess the ability to reduce Cu(II) to Cu(I).

Fig. 3. Detection of A) L-DOPA and B) DA induced Cu (I) production by neocuproine.

Fig. 3

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Reaction mixture (3.0 ml) contained 10 mM Tris-HCl (pH 7.5) along with 300 μM neocuproine and indicated concentrations of the following components: 1) Neocuproine + 100 μM Cu(II), 2) Neocuproine + 50 μM L-DOPA/DA, 3) Neocuproine + 50 μM L-DOPA/DA + 100 μM Cu(II).

3.4. Molecular docking

As obvious from results (Fig. 4), L-DOPA forms 7 hydrogen bonds with nitrogenous bases of B-DNA (A-3, A-4, C-11, T-5 and G-10) resulting in a more negative AutoDock binding energy (−7.01 kcal/mol) and a lesser AutoDock inhibition constant (7.24 μM). As shown in Fig. 5, DA forms only 5 hydrogen bonds with B-DNA (A-6, A-7, T-5 and T-6) as compared to L-DOPA. This decreased hydrogen bonding lead to a lesser negative binding energy (−6.67 kcal/mol) and a higher inhibition constant (16.09 μM) Table1. Even though DA possesses same number of hydroxyl groups (2) as L-DOPA, it tends to form only 5 hydrogen bonds with B-DNA. The additional hydrogen bonding of L-DOPA with B-DNA can be attributed to the structural aspects of this compound. The presence of a carboxylic group in L-DOPA seems to facilitate additional interaction with DNA via hydrogen bonding which might provide higher stability. However, formation of only 5 hydrogen bonds with nitrogenous bases (A-6, A-7, T-5 and T-6) of B-DNA validates the lesser stability of DA with B-DNA. Based on these results, it can be inferred that L-DOPA has a relatively higher affinity for DNA as compared to DA.

Fig. 4. Molecular docked structure of L-DOPA complexed with B-DNA.

Fig. 4

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(A) Surface view interaction of L-DOPA with B-DNA. (B) Hydrogen bonding interactions of L-DOPA with dodecamer duplex of the sequence (CGCGAATTCGCG)2 (PDB ID: 1BNA).

Fig. 5. Molecular docked structure of dopamine complexed with B-DNA.

Fig. 5

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(A) Surface view interaction of dopamine with B-DNA. (B) Hydrogen bonding interactions of dopamine with dodecamer duplex of the sequence (CGCGAATTCGCG)2 (PDB ID: 1BNA).

Table 1.

Hydrogen bonds, inhibition constant and binding energy as obtained by AutoDock results of two docked ligands.

Ligand AutoDock Binding Energy (Kcal/mol) AutoDock Inhibition Constant (μM) No. of Hydrogen bonds
L-DOPA −7.01 7.24 7
DOPAMINE −6.67 16.09 5
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3.5. Agarose gel electrophoresis of plasmid DNA treated with compounds and Cu (II)

As an initial approach to determine the efficacy of L-DOPA/DA- Cu(II) complex in cleaving DNA, plasmid pcDNA3.1 (+/−) was treated with L-DOPA and DA in the presence of copper. As depicted in Fig. 6, the addition of copper to the compounds, L-DOPA/DA, resulted in the generation of open circular and linear topological forms of plasmid DNA. The conversion of plasmid DNA into open circular form by the compounds was observed even at very low (~1 μM) concentration of copper (Supplementary Fig. S1). At higher concentrations, DNA appeared as smear of fragments, indicative of extensive DNA damage. Fig. 7 shows the effect of increasing concentrations of compounds leads to production of open circular and linear forms of the plasmid in the presence of Cu(II). However, under identical conditions, L-DOPA, DA or copper alone did not result in substantial DNA breakage. It was of further interest to determine the effect of selective copper chelators-neocuproine, bathocuproine and non-specific metal chelator, EDTA on such DNA strand breakage. As shown in Fig. 8, addition of these chelators abolished the DNA breakage caused by L-DOPA/DA- Cu(II) system. The results imply that Cu(I) is the intermediate formed upon the interaction of compounds with Cu(II) and is responsible for causing DNA strand breakage.

Fig. 6. Agarose gel electrophoretic pattern of ethidium bromide stained plasmid pcDNA3.1 (+/−) after treatment with increasing concentrations of Cu(II) in the presence of compounds A) L-DOPA and B) DA.

Fig. 6

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Lane a: Linear DNA marker; Lane b: DNA alone; Lane c: DNA + L-DOPA/DA (60μM); Lane d: DNA + L-DOPA/DA (60μM) + Cu(II) (1.25 μM); Lane e: DNA + L-DOPA/DA (60μM)+ Cu(II) (2.5μM); Lane f: DNA + L-DOPA/DA (60μM) + Cu(II) (5 μM); Lane g: DNA + L-DOPA/DA (60μM) + Cu(II) (10 μM); Lane h: DNA + L-DOPA/DA (60μM) + Cu(II) (15μM); Lane i: DNA + L-DOPA/DA (60μM) + Cu(II) (20μM); Lane j: DNA + L-DOPA/DA (60μM) + Cu(II) (25μM).

Fig. 7. Agarose gel electrophoretic pattern of ethidium bromide stained plasmid pcDNA3.1 (+/−) after treatment with increasing concentrations of compounds A) L-DOPA and B) DA in the presence of Cu(II).

Fig. 7

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Lane a: Linear DNA marker; Lane b: DNA alone; Lane c: DNA + Cu(II) (5μM); Lane d: DNA + L-DOPA/DA (1.23μM) + Cu(II) (5 μM); Lane e: DNA + L-DOPA/DA (3.7μM)+ Cu(II) (5μM); Lane f: DNA + L-DOPA/DA (11.1μM) + Cu(II) (5 μM); Lane g: DNA + L-DOPA/DA (33.3μM) + Cu(II) (5μM); Lane h: DNA + L-DOPA/DA (100μM) + Cu(II) (5μM); Lane i: DNA + L-DOPA/DA (300μM) + Cu(II) (5μM).

Fig. 8. Agarose gel electrophoretic pattern of ethidium bromide stained plasmid pcDNA3.1 (+/−) treated with compounds A) L-DOPA, B) DA and Cu(II) in the presence of copper chelators.

Fig. 8

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Lane a: DNA alone; Lane b: DNA + Cu(II) (5 μM); Lane c: DNA + L-DOPA/DA (50μM); Lane d: DNA + L-DOPA/DA (50μM) + Cu(II) (5μM); Lane e: DNA + L-DOPA/DA (50μM) + Cu(II) (5μM) + Bathocuproine (1mM); Lane f: DNA + L-DOPA/DA (50μM) + Cu(II) (5μM) + Neocuproine (1mM); Lane g: DNA + L-DOPA/DA (50μM) + Cu(II) (5μM) + EDTA (1mM).

3.6. Effect of copper chelating agent neocuproine on L-DOPA/DA induced DNA damage in human lymphocytes as measured by Comet assay

In the present study, increasing concentrations of L-DOPA and DA were tested for their ability to induce cellular DNA breakage in human lymphocytes using the comet assay (Fig. 9). A dose dependent increase in the DNA breakage induced by L-DOPA and DA is evidenced by increasing comet tail lengths indicative of significant breakage of cellular DNA. Since there was no added copper to the reaction, we take these results to imply that L-DOPA and DA cause oxidative DNA breakage by directly mobilizing endogenous chromatin-bound copper. Further, a cell membrane permeable Cu(I) specific chelator (neocuproine) was used to study its effect on DNA breakage induced by L-DOPA or DA under identical conditions. A significant inhibition of L-DOPA or DA induced DNA degradation was observed in the presence of neocuproine as evidenced by the decreased Comet tail lengths. A slightly higher level of DNA breakage is observed with L-DOPA (Fig. 9A) as compared to DA (Fig. 9B), which might be attributed to the higher DNA binding affinity of L-DOPA.

Fig. 9. Effect of copper chelating agent neocuproine on DNA breakage induced by A) L-DOPA and B) DA in human peripheral lymphocytes.

Fig. 9

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Comet tail length (μm) was plotted as a function of increasing concentrations of compounds (0–400 μM) in the absence and presence of (50 μM) Neocuproine. All points represent mean of three independent experiments. Error bars denote Mean ± SEM. **p < 0.05 when compared with *(untreated control cells).

3.7. Induction of Double Strand Breaks (DSBs) by L-DOPA/DA in cancer cell lines

Double strand DNA breaks are considered as the most genotoxic DNA lesions and if unrepaired, can lead to diseases (Thanan et al., 2014). Following cleavage of both the strands of DNA, Ser139 on the C-terminal of histone H2AX is known to be immensely and rapidly phosphorylated (Rogakou et al., 1998). This phosphorylated histone, referred to as γH2AX, is considered to be robust and reliable marker for double strand breaks (Fernandez-Capetillo et al., 2004; Sedelnikova et al., 2002). In order to evaluate the effect of L-DOPA/DA on DNA integrity, the DNA damage inducing capability of these compounds was detected by the formation of γH2AX. Gli36 and HCT116 cancer cells were treated with increasing concentrations (5–500 μM) of the compounds (L-DOPA and DA) and harvested after 1 hr. Normal skin fibroblasts (OSU2) were analyzed similarly for comparison with cancer cells. Whole cell lysates were subjected to western blotting to determine the induction of γH2AX. Fig. 10 and Fig. 11 demonstrate that L-DOPA and DA induce a significant γH2AX formation in Gli36 and HCT116 cells. However, it is noteworthy that a slightly higher level of γH2AX induction is observed in HCT116 cells as compared to Gli36 cells with both the compounds. Interestingly, HCT116 cells have been reported to accumulate higher levels of copper (~7 fold) than normal colon cells (Fatfat et al., 2014). In contrast, no γH2AX was observed in normal skin fibroblasts (OSU2) with either of the compounds (Fig. 10A and Fig. 11A).

Fig. 10. L-DOPA induces γH2AX in Gli36 and HCT116 cells but not in OSU2.

Fig. 10

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Representative blots of OSU2 (A), Gli36 (B) and HCT116 (C) cells treated with increasing concentrations of L-DOPA (5, 10, 50, 500 μM respectively) and harvested after 1 hour. Whole cell lysates were immunoblotted for the DSB marker, γH2AX.

Fig. 11. DA induces γH2AX in Gli36 and HCT116 cells but not in OSU2.

Fig. 11

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Representative blots of OSU2 (A), Gli36 (B) and HCT116 (C) cells treated with increasing concentrations of DA (5, 10, 50, 500 μM respectively) and harvested after 1 hour. Whole cell lysates were immunoblotted for the DSB marker, γH2AX.

Apparently, there is no induction of γH2AX even at 500 μM concentration of the compounds in OSU2, whereas there is an obvious γH2AX formation in Gli36 and HCT116 at 50 μM concentration of the compounds. The results imply that the DSBs inducing capability of these catecholamines correlates with the inherent level of copper in the cells. These data suggest that the compounds have selectivity for inducing DNA damage in cancer cells. Further, the reason for this observation can be attributed to the elevated levels of copper in cancer cells, which render them susceptible to DNA damage. Hence, these observations suggest that L-DOPA and DA can incur significant DNA damage by mobilizing endogenous redox active copper.

3.8. Detection of L-DOPA/DA induced γH2AX and 53BP1 foci in the absence and presence of copper

Upon introduction of genotoxic damage in cells, DNA Damage Response (DDR) induces recruitment of specific damage sensing and repair factors into distinct foci at damage sites. The phosphorylation of variant H2A (γH2AX) is known to be one of the earliest events during this orchestrated response (Jackson and Bartek, 2009). In human cells, γH2AX spans ~2Mb DNA region and forms foci that can be detected by immunofluorescence microscopy (Rogakou et al., 1998). γH2AX, in turn, facilitates efficient retention of downstream repair factors such as BRCA1 and 53BP1 at the damage sites (Sharma et al., 2014). In this experiment, we investigated the induction and co-localization of γH2AX and 53BP1 foci in Gli36 cells at the damage sites. For this, Gli36 cells were subjected to treatment with compounds, L-DOPA and DA in the absence and presence of copper in serum free medium for 1 hr (Fig. 12).

Similarly, the cells were treated with phleomycin, which was used as a positive control for this experiment. It is observed that the foci formation is enhanced when cells are treated with L-DOPA/DA in the presence of copper. However, copper, L-DOPA or DA alone do not lead to significant foci formation under identical conditions. Further, as implied from previous results, L-DOPA (Fig. 12A) is found to be more efficient in inducing DNA damage as compared to DA (Fig. 12B).

4. Discussion

Endogenous metabolites have been implicated in oxidative DNA damage leading to mutagenesis, carcinogenesis and ageing (Rehmani and Hadi, 2015). Evaluation of in vitro DNA damage by endogenous compounds is relevant in providing a putative overview of their in vivo toxicity. Since the detection of DNA adducts derived from endogenous electrophiles, the contribution of endogenous genotoxins in carcinogenesis has emerged as an important field of research. Elevated levels of oxidative damage has also been shown to be inversely proportional to life span and lead to several disease conditions (Spencer et al., 2011). The main neuropathological feature of PD is the loss of dopaminergic neurons in the substantia nigra of brain, which having oxidizable metabolites and relatively low antioxidant complement is highly susceptible to oxidative damage (Gaggelli et al., 2006; Jayaraj et al., 2012).

The oxidation of catechol neurotransmitters is known to proceed non-enzymatically in the presence of transition metal ions (Lévay et al., 1997; Spencer et al., 1994). Further, catechol neurotransmitters possess the ability to redox cycle, generating DNA-damaging ROS (Lévay et al., 1997; Manini et al., 2007; Masserano et al., 1999; Miyazaki and Asanuma, 2008). Copper is prevalent in human tissues and its elevated levels have been identified in brain tissues of patients with neurodegeneration (Dexter et al., 1991; Spencer et al., 1994). In a recent study, copper levels in normal brain tissue were found to be approximately 16 and 31 ng/g in the substantia nigra and the locus coeruleus, respectively (Zecca et al., 2004). Moreover, cancer cells possess the tendency to accumulate high levels of copper, a characteristic feature observed in breast, colorectal, leukemia and ovarian cancer (Gupte and Mumper, 2009) and may involve in electron transfer with catecholamines to induce DNA damage. Some studies have suggested that occupational exposures to manganese and copper, in particular, are risk factors for neurological disease conditions such as Parkinson’s disease (Gorell et al., 1997). As suggested by Spencer et al. (2011), the presence of redox active catecholamine neurotransmitters along with the localization of catalytic copper to DNA implies a possible role of these agents in oxidatively generated DNA damage.

The present study clearly demonstrates that catecholamines (L-DOPA and DA) can induce significant DNA damage by mobilizing redox active metals such as copper. The results of fluorescence spectroscopy and molecular docking indicate that L-DOPA and to a lesser extent, DA is capable to bind with DNA and Cu(II). Further, we have shown that these catecholamines possess the ability to reduce Cu(II) to Cu(I) and might be involved in the redox cycling of copper. As evident from plasmid nicking and comet assays, the addition of copper specific chelators leads to diminution of DNA damage, suggesting the role of Cu(I) as an essential intermediate in the DNA breakage reaction. Further, the induction of DSB marker, γH2AX was found to be more pronounced in cancer cells (Gli36 and HCT116) which are known to accumulate high levels of copper. The foci formation and co-localization of DNA repair factors γH2AX and 53BP1 at damage sites appears to be enhanced in the presence of copper. Taken together, these results indicate copper binding as a primary factor for the observed pro-oxidant DNA damage by the catecholamines. Although these compounds have the same mechanism of action, structural differences resulted in differential ability to induce DNA damage. Hence, L-DOPA is more efficient as a pro-oxidant than DA, possibly due to its relatively higher binding affinity towards DNA and Cu(II). Further, this study substantiates copper dependent enhancement of DNA damage as observed with several catechol compounds (Fazal et al., 1990; Husain and Hadi, 1998; Li et al., 1994). We would like to emphasize that similar to many antioxidants of plant and animal origin, endogenous catecholamines might act as pro-oxidants or antioxidants based on the physiological concentration (Asad et al., 1999; Bhat and Hadi, 1994).

Based on our findings, we postulate that these catecholamines form a ternary complex with DNA and Cu(II), facilitating redox cycling of copper and generating ROS similar to various DNA cleavage systems (Hadi et al., 2007). The production of ROS by such mechanism is likely to proceed by Haber-Weiss or Fenton reactions, which are capable of damaging biological macromolecules such as DNA (Halliwell and Gutteridge, 1990). Although the concentrations of Cu(II) employed in this study seem to be relatively high, the copper concentrations in various pathophysiological disorders such as PD and Wilson’s disease, are significantly elevated to potentiate DNA damage (Spencer et al., 1994). It is noteworthy that labile/non-protein bound copper concentrations in the brains of Alzheimer’s patients are approximately seven times higher than normal (Squitti et al., 2006). Further, the concentration of catecholamines employed in this study for inducing DNA damage are higher than in vivo levels (Garcı´a et al., 2012) and the therapeutic plasma concentrations in PD patients (Rossor et al., 1980). However, Parkinsonian patients, chronically exposed to L-DOPA therapy, are potentially at risk from the toxic intermediates of the catecholamines. Since DNA breakage by other metabolites is documented (Rehmani and Hadi, 2015), the cumulative effect of various endogenous metabolites may be physiologically significant and could contribute to the etiology of genetic diseases such as cancer and neurological diseases.

5. Conclusion

In conclusion, we present a pro-oxidant mechanism providing insights into the pathogenesis of diseases such as neurological disorders and cancer. This is an alternative, non-enzymatic and copper-mediated pathway that involves mobilization of endogenous copper. Overall, we suggest that the putative mechanism can serve as a basis for formulating novel chemopreventive and treatment strategies against oxidative DNA damage mediated diseases. The outcomes of this study are clinically relevant as the putative toxicity of catecholamines might be attenuated by naturally occurring antioxidants or metal chelators. Drugs or compounds designed to interfere with metal-endogenous metabolite interactions might comprise a novel approach for treating neurodegenerative diseases. Future research in this realm should focus on elucidating implications and relevance for toxic effects exerted by endogenous genotoxins in vivo. Such studies could open promising new avenues for ameliorating the pathogenesis and deleterious effects of DNA-damage mediated disease conditions, especially neurodegeneration and cancer.

Highlights.

Catecholamines induce significant DNA damage by mobilizing endogenous Cu(II) ions.

L-DOPA and dopamine possess the ability to reduce Cu(II) to Cu(I).

L-DOPA and dopamine induce Cu(II)-mediated foci formation and co-localization of γH2AX & 53BP1 at damage sites.

Owing to its structural differences, L-DOPA is more efficient as a pro-oxidant than dopamine.

Acknowledgments

Nida Rehmani was an Indo-US 21st Century Obama-Singh Knowledge Initiative (STEM-ER) Fellow at the Ohio State University and her research effort was supported by NIEHS grants ES2388 and ES12991 (to AAW).

Footnotes

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Conflict of interest

The authors disclose no conflict of interest.

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