ParaquatâMelanin Redox-Cycling: Evidence from Electrochemical

Subscriber access provided by UNIV OF CAMBRIDGE

Article

Paraquat-Melanin Redox-Cycling: Evidence from Electrochemical Reverse Engineering Eunkyoung Kim, W. Taylor Leverage, Yi Liu, Lucia Panzella, Maria Laura Alfieri, Alessandra Napolitano, William E Bentley, and Gregory F. Payne ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00007 • Publication Date (Web): 01 Jun 2016 Downloaded from http://pubs.acs.org on June 5, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

Paraquat-Melanin Redox-Cycling: Evidence from Electrochemical Reverse Engineering Eunkyoung Kim1,2, W. Taylor Leverage1,2, Yi Liu1,2, Lucia Panzella3, Maria Laura Alfieri3, Alessandra Napolitano3, William E. Bentley1,2 and Gregory F. Payne1,2

1. Institute for Bioscience and Biotechnology Research, University of Maryland 5115 Plant Sciences Building College Park, MD 20742, USA 2. Fischell Department of Bioengineering University of Maryland, College Park, MD 20742, USA 3. Department of Chemical Sciences, University of Naples Federico II Via Cintia 4, I-80126 Naples (Italy)

* Corresponding author Email: [email protected] Phone: 301-405-8389 FAX: 301-314-9075

ABSTRACT Parkinson’s disease is a neurodegenerative disorder associated with oxidative stress and the death of melanin-containing neurons of the substantia nigra. Epidemiological evidence links exposure to the pesticide paraquat (PQ) to Parkinson’s disease and this link has been explained by a redox-cycling mechanism that induces oxidative stress. Here, we used a novel electrochemistry-based reverse engineering methodology to test the hypothesis that PQ can undergo reductive redox-cycling with melanin. In this method, (i) an insoluble natural melanin (from Sepia melanin) and a synthetic model 1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

melanin (having a cysteinyldopamine-melanin core and dopamine-melanin shell) were entrapped in a non-conducting hydrogel film adjacent to an electrode, (ii) the film-coated electrode was immersed in solutions containing PQ (putative redox-cycling reductant) and a redox-cycling oxidant (ferrocene dimethanol), (iii) sequences of input potentials (i.e., voltages) were imposed to the underlying electrode to systematically engage reductive and oxidative redox-cycling, and (iv) output response currents were analyzed for signatures of redox-cycling. The response characteristics of the PQ-melanin systems to various input potential sequences support the hypothesis that PQ can directly donate electrons to melanin. This observation of PQ-melanin redox-interactions demonstrates an association between two components that have been individually linked to oxidative stress and Parkinson’s disease. Potentially, melanin’s redox-activity could be an important component in understanding the etiology of neurological disorders such as Parkinson’s disease. Keywords: Electrochemistry, Melanin, Oxidative Stress, Paraquat, Redox-cycling, Reverse Engineering

INTRODUCTION Strong epidemiological evidence links exposure to the pesticide paraquat (PQ) to the development of Parkinson’s disease.1-3 Paraquat is believed to act by increasing oxidative stress4, 5 through the putative redox-cycling mechanism illustrated in Scheme 1a which shows both the depletion of cellular reducing equivalents and the generation of damaging reactive oxygen species (ROS).6, 7 Thermodynamically, redox-cycling would seem an anomaly – how could a molecule exist in conditions that simultaneously favor reduction and oxidation? Biological contexts that are characterized by steep O2 gradients provide such conditions since molecules with redox-cycling capabilities only need to transverse small distances to switch between exposure to conditions that favor reduction and those that favor oxidation. Specifically, steep O2 gradients can occur in metabolically active tissue (e.g., the brain) where the O2 supplied from capillaries is consumed over small (tens of microns) distances.8, 9 The neuromelanin pigment has also been suggested to play a role in the development of Parkinson’s disease.10-13 This pigment is present at high levels in the dopaminergic neurons of the substantia nigra which are selectively vulnerable and degenerate over the course of Parkinson’s disease.10 Both the pathway to melanin biosynthesis and the melanin pigment itself have been reported to have abilities to either promote or protect against oxidative stress.14-16 It has been suggested that the biosynthesis of melanin can contribute to oxidative stress through the generation of the reactive o-quinone intermediates and ROS byproducts,6, 17 but it has also been argued that melanin synthesis could also offer protection by sequestering the reactive quinones in an insoluble form.13, 18 The melanin pigment itself has been reported to have context-dependent effects; offering protection by scavenging free radicals and chelating metals (e.g., iron), but contributing to oxidative stress through the release of insoluble melanin upon neuronal cell death19 (stress may result from the depletion of cellular antioxidants20 or through neuroinflammatory pathways10). Electrochemistry is emerging as an in vitro tool for investigations in redox biology since it permits highly sensitive measurements of oxidation and reduction reactions.21 For instance, electrochemical methods have been investigated to: evaluate the antioxidant activities of food phenolics;22-25 mimic oxidative drug metabolism (e.g., phase I P450 enzymes);26-31 and correlate the biological activities of bioreductive 2 ACS Paragon Plus Environment

Page 2 of 25

Page 3 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


antitumor drugs to their redox potentials.32 In addition, electrochemistry allows arbitrarily complex potential inputs to be imposed while the output currents are in a convenient format for analysis by advanced algorithms (e.g., signal processing). For instance, the analysis of complex electrochemical inputs and outputs has been used to: discern concentrations of key metabolites (e.g., glucose) by dynamic electrochemistry;33-35 detect biologically-relevant chemical activities (e.g., redox-cycling);36 and reverse engineer the redox-properties of biological materials.37-39 Thus, electrochemistry offers a set of measurement capabilities that complement chemical and biological methods for probing the subtle interactions of redox-biology.40, 41 Previously, we developed electrochemical methods to study context-dependent redox-reactions with catecholic materials (e.g., melanins).42, 43 To study melanin, Scheme 1b shows that we localize the insoluble melanin sample adjacent to an electrode surface by entrapping it within a film of the aminopolysaccharide chitosan. Neuromelanins are generally unavailable, and thus model melanins must be selected for investigation.44 We selected the natural melanin from cuttlefish (Sepia melanin) because: it is commercially available; it is composed of eumelanin which is the primary18, 45-47 and surface-exposed component of neuromelanin; and it has been used as a model to investigate solutemelanin interactions (e.g., for metal binding).44, 48, 49 We also investigated a synthetic model with a cysteinyldopamine-derived core and a dopamine-derived surface coating which mimics the structure of neuromelanin.19, 47, 50 By cycling the imposed potential (voltage) input to the underlying electrode, it is possible to switch between oxidizing and reducing contexts. Importantly, because of melanin’s insolubility, we use soluble mediators to shuttle electrons between the underlying electrode and the entrapped melanin. These mediators “transmit” the imposed redox context to the entrapped melanin. In essence, the in vitro temporal cycling of imposed redox conditions provides a simple means to mimic biologically-relevant spatial gradients in redox conditions. Using this method, we previously demonstrated that insoluble melanin retains redox-activity and can accept electrons from reductants and donate electrons to O2 to generate ROS.43 Here, we extend these observations to demonstrate that melanin can directly engage in redox-cycling by accepting electrons from PQ. RESULTS AND DISCUSSION Initial Studies for Electrochemical Reverse Engineering with Sepia Melanin Figure 1a illustrates the basis for this electrochemical reverse engineering method for detecting redoxinteractions with the insoluble melanin. Under reducing conditions, a soluble shuttle undergoes reductive redox-cycling that serves to transfer electrons from the electrode to the melanin. The hypothesis of this work is that PQ can engage melanin in such reductive redox-cycling reactions. A second shuttle is used to engage the melanin in oxidative redox-cycling to transfer electrons from the melanin to the electrode. Ferrocene dimethanol (Fc) is a convenient oxidative redox-cycling compound because of its reversible redox activity.51 These reductive and oxidative redox-cycling reactions can be sequenced by cycling the imposed electrode potential between reducing and oxidizing voltages. A prerequisite for testing melanin’s ability to undergo redox-cycling is illustrated in the thermodynamic plot of Figure 1a: the redox potentials of the two electron shuttles must bracket the redox potential of 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

melanin which for Sepia melanin has been estimated to range from -0.2 V to +0.25 V (vs Ag/AgCl).43 To evaluate the intrinsic redox potentials for PQ (putative reductive redox-cycler) and Fc (oxidative redoxcycler), we performed cyclic voltammetry (CV) measurements with gold electrode (no films) by imposing the cyclic potential between +0.5 V and -0.8 V (vs Ag/AgCl). For the first control, a gold electrode was immersed in phosphate buffer (0.1 M, pH 7.0) without redox-active shuttles. The cyclic voltammogram (CV) in Figure 1b for this buffer control shows no oxidation peaks while a sharp reductive peak was observed as the potential was scanned toward -0.8 V. This reductive peak is due to water electrolysis which interferes with measurements and limits the opportunity to interpret results in this reducing region. The second control is phosphate buffer containing PQ (50 µM) and the CV for this PQ control in Figure 1b shows no peak currents under oxidative conditions but a shoulder reductive peak at -0.65 V. This reductive peak is consistent with previous reports that PQ undergoes electrochemical reduction at 0.63 V (vs Ag/AgCl).52 The third control is phosphate buffer containing Fc (50 μM) and Figure 1c shows the result for this control. As expected, the CV for this Fc control shows strong oxidative and reductive peaks between +0.2 and +0.3 V which is consistent with Fc’s redox potential (E° = +0.25 V vs Ag/AgCl).21, 53 In summary, Figure 1 shows that Fc and PQ are each redox-active and have redox potentials that bracket the redox potential reported for Sepia melanin.43, 54 To provide initial evidence that PQ can redox-cycle with Sepia melanin, we compared the response of a melanin-containing chitosan film to responses from two control film-coated electrodes. These three film-coated electrodes were individually immersed in solutions containing both PQ (50 µM) and Fc (50 µM) and the potential cycled between +0.5 V and -0.8 V (vs. Ag/AgCl). The negative control is redoxinactive chitosan film and Figure 2 shows small peaks for PQ reduction and Fc oxidation. These peaks are attributed to diffusion of PQ and Fc from the solution through the chitosan film to the electrode. A positive control is a chitosan film that had been modified with catechols. Catechol-modified chitosan films have been observed to be redox-active with a redox potential similar to that of Sepia melanin and these films have been observed to readily exchange electrons with a wide range of oxidants and reductants.21, 42, 53 In Figure 2, the catechol-chitosan films showed that large oxidation currents of Fc are observed when the potential is cycled to positive voltages and large reducing currents are observed when the potential is cycled to negative voltages. The high amplification of paired redox currents is due to two redox–cycling reactions as described in Figure 1a. Under reducing imposed potentials, reductive redox-cycling can occur (top in Figure 1a), such that PQ2+ diffuses through film, is reduced to PQ+• at the electrode and can be re-oxidized by donating its electron to oxidized catechol-chitosan film. Under oxidizing imposed potentials, oxidative-redox-cycling can occur (bottom in Figure 1a), such that Fc is oxidized to Fc+ by donating electrons to the electrode and is re-reduced by accepting an electron from the reduced catechol-chitosan. These two redox-cycling reactions can cause the paired amplification of Fc and PQ currents for catechol-chitosan, which is an electrochemical signature indicating that the modified film is redox-active and can reversibly interact with two electron shuttling molecules through redox-cycling reaction. Figure 2 shows the results for the electrode coated with the melanin-containing chitosan film are intermediate between those of the positive and negative controls. Compared to the negative control, 4 ACS Paragon Plus Environment

Page 4 of 25

Page 5 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the melanin-chitosan films show amplified Fc oxidation currents and PQ reduction currents. This paired amplification is consistent with previous results and demonstrates that Sepia melanin confers redox activity to the film.43 Compared to the positive control, the melanin-chitosan film has a lower redox activity, presumably because the particulate nature of this melanin imposes a mass transfer barrier for the soluble electron shuttles to access the redox-active moieties.43 These results provide initial evidence that PQ can serve as a shuttle to transfer electrons between the electrode and Sepia melanin. Cyclic Potential Input with Sepia Melanin To provide more rigorous evidence for PQ-melanin redox-cycling, we subjected the melanin-containing films to repeated cyclic potential inputs to determine the stability of the redox-cycling signatures. Specifically, we prepared electrodes coated with melanin-chitosan and chitosan (control) films, and immersed these films in buffered solutions containing both PQ (50 μM) and Fc (50 μM) and the imposed potential was cycled between +0.5 V and -0.8 V for 20 cycles (scan rate 2 mV/s). Figure 3a shows the CVs for these two film-coated electrodes. The CVs for the control chitosan film shows small peaks for Fc oxidation and reduction (E° = +0.25 V vs Ag/AgCl), and the shoulder peak for PQ reduction at -0.70 V. The CV for the film with entrapped Sepia melanin shows considerable amplification of both PQ’s reduction and Fc’s oxidation and after the first cycle, there were no noticeable changes in the output currents over 20 repeated cycles. (The steady redox current outputs of catechol-chitosan film were also observed in the catechol-chitosan film as shown in Figure S1 of Supporting Information.) The results in Figure 3a are re-plotted as input-output curves in Figure 3b. Visually, it appears from this plot that the amplified PQ reduction and Fc oxidation for the melanin-containing film were “steady” over multiple cycles with little attenuation in peak currents. To support this visual observation, we analyzed the individual “signals” from the CVs as illustrated in Figure 3c. Specifically, the CVs are divided into 4 quadrants based on whether the currents are oxidizing or reducing, and whether the currents are attributed to Fc or PQ. It is important to note, that the assignment of output currents to a single mediator is used for data analysis and is only an approximation of the underlying electrochemical reactions. The current for each quadrant is integrated to generate the charge transfer (Q) as illustrated in Figure 3c. The calculated Q values for the Fc region are shown in Figure 3d while the calculated Q values for the PQ region are shown in Figure 3e. There are two important observations from Figure 3. First, Figure 3d shows a greater Fc-oxidative charge transfer for the melanin-chitosan film compared to the control chitosan film. Similarly, Figure 3e shows a greater PQ-reductive charge transfer for the melanin-chitosan film compared to the control chitosan film. This “excess” electron transfer is quantified by redox capacity, NOx and NRed, as illustrated by the equations in Figure 3c, and Figure 3f plots NOx and NRed as a function of cycle number. Averaging over the various cycles yielded values of 21 ± 0.5 nmole/cm2 for Fc’s NOx and 15 ± 0.4 nmole/cm2 for PQ’s NRed. This amplification of Fc’s oxidation and PQ’s reduction in the presence of Sepia melanin is consistent with the redox-cycling reactions of Figure 1a. The second observation in Figure 3 is that the output Q’s, NOx or NRed appear approximately constant (i.e., steady) over the various cycles. [Note: the gradual changes observed over this 8 hour experiment are possibly due to small amounts of evaporation.] A steady amplification of Fc-oxidation and PQ-reduction indicates that Sepia melanin 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

undergoes repeated redox-cycling: that is, melanin is repeatedly and reversibly oxidized and reduced under these experimental conditions. (Further analysis of Figure 3 is provided in Figure S2 of Supporting Information) An additional test involved the generation of CVs from buffered solutions containing PQ (50 μM) but not Fc. As expected, Figure 4a and Figure 4b show no Fc oxidation peaks for either the melanin-chitosan film or the control chitosan film. When the solution contains PQ but not Fc, the entrapped Sepia melanin can be “charged” by PQ’s reductive redox-cycling, however, the electrons transferred to melanin cannot be “discharged”. Because of the finite capacity of melanin to accept electrons, the CVs and input-output curves in Figure 4a and Figure 4b show the reductive currents are non-steady but rather decrease monotonically with each cycle. Figure 4c illustrates this non-steady output by contrasting the decay in PQ’s NRed in the absence of Fc, to the steady output (constant NRed) observed in the presence of both Fc and PQ. (Further analysis of Figure 4 is provided in Figure S3 of Supporting Information.) In sum, Figure 3 shows paired amplifications in oxidative and reductive currents and steady output when Sepia melanin was probed using cyclic potential inputs with both Fc and PQ. In the absence of Fc (Figure 4), non-steady output responses were observed in the PQ-melanin interactions. These observations support the hypothesis that PQ donates electrons to melanin through reductive redox-cycling reactions. Step Potential Inputs with Sepia Melanin In the next set of experiments, film-coated electrodes were probed in the presence of both Fc (50 μM) and PQ (50 μM) using step potential inputs as illustrated in Figure 5a. The initial reducing condition (0.65 V vs Ag/AgCl) was used to engage PQ in reductive redox-cycling to transfer electrons from the electrode to the entrapped melanin. After this initial charging step, the potential was stepped to an oxidative potential (+0.5 V) to engage Fc’s redox-cycling to discharge the melanin-containing film. Figure 5b shows that the charge transfer (Q) during this 30 minute discharging step was monitored and compared against a control electrode coated with chitosan. The results and calculations in Figure 5b show that during this 30 minute discharging step, a considerable excess electron transfer from the melanin-chitosan film was observed (NOx = 172 ± 8 nmol/cm2). This value of NOx is 8-fold larger than that observed in Figure 3f, presumably because a longer time was used to discharge electrons from the melanin during this prolonged oxidation. The final step in Figure 5a is to undergo reducing conditions that allow a re-charging of the melanin through the PQ-mediated reductive redox-cycling mechanism. In this step, we employed a relatively mild reducing potential (-0.65 V) relative to PQ’s redox potential (E° ≈ - 0.63 V) in order to minimize interference from the electrolysis of water (e.g., see Figure 1b). Figure 5c shows that the electron transfer to the melanin-containing film (vs the control chitosan film) during the reducing step (NRed) is 83 ± 20 nmole/cm2. The relatively large error bars for NRed (measurements performed in triplicate) are indicative of the high background under these reducing conditions (e.g., high noise). The comparatively lower NRed (vs NOx) presumably reflects the milder conditions (i.e., lower over-potentials) used for reductive redox-cycling (vs oxidative redox-cycling). [Note: over-potential refers to the imposed 6 ACS Paragon Plus Environment

Page 6 of 25

Page 7 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


potential vs E° and provides a measure of the thermodynamic driving force for electron transfer (+0.5 − +0.25 = 0.25 V for Fc-oxidation; and -0.65 – (-0.63) = -0.02 V for PQ-reduction)]. In sum, the responses observed in Figure 5 from the step potential inputs provides further evidence that PQ’s reductive redox-cycling provides a mechanism to transfer electrons from the electrode to insoluble Sepia melanin. Cyclic Potential Inputs with Step Increases in Amplitude with Sepia Melanin In the final test for redox-cycling, film-coated electrodes were probed in solutions containing both Fc (50 μM) and PQ (50 μM) using the cyclic potential inputs illustrated in Figure 6a. Initially, the input amplitude (i.e., the potential range) was sequenced between +0.5 and -0.4 V (vs Ag/AgCl) which is sufficiently oxidative for Fc to be oxidized to Fc+. However, this initial input amplitude is insufficiently reductive to allow PQ to be reduced. As expected, the CV in Figure 6b (3rd cycle) for this initial amplitude shows redox peaks near +0.3 V for Fc, while no reducing peaks are observed at more negative potentials. Over time, Figure 6a shows the potential amplitude was expanded to reducing conditions sufficient to allow PQ reduction. The final three CVs in Figure 6b show that as this input amplitude is progressively expanded to include reducing conditions, output currents emerge associated with PQ’s reduction. This observation is expected: perturbations in input reducing conditions (that allow PQ reduction) should result in perturbations in output reducing currents associated with PQ reduction. Further, this observation is expected in the presence or absence of redox-cycling interactions and thus both the melanin-chitosan and control chitosan films display this response. The novel feature of this methodology is that if redox-cycling occurs, then perturbations in input reducing conditions (that allow PQ reduction) result in perturbations in output oxidative currents associated with Fc oxidation. Thus, redox-cycling links a perturbation in PQ reduction to a response in Fc oxidation. Specifically, if melanin redox-cycles with both PQ and Fc, then conditions that limit PQ’s reductive redox-cycling should also limit Fc’s redox-cycling, while perturbations that allow PQ’s reductive redox-cycling should also allow Fc’s oxidative redox-cycling. The input-output curves in Figure 6a and the sequence of CV’s in Figure 6b show that as the input potential amplitude is progressively expanded into the reducing region, then progressively larger output currents are observed in the oxidative region associated with Fc oxidation. Mechanistically, the observed behavior is explained by melanin’s finite redox capacity. The initial input potential that allows Fc oxidative redox-cycling (but not PQ reductive redox-cycling) serves to oxidize the melanin and deplete it of electrons. If the melanin is depleted of electrons, Fc can no longer undergo oxidative redox-cycling and the associated oxidative currents are suppressed. However, as the potential amplitude is expanded into reducing conditions that allow PQ to redox-cycle, then the melanin can be re-charged with electrons and this PQ-mediated re-charging allows Fc to engage in oxidative redoxcycling. Using the same analysis as in Figure 3, Figure 6c shows the calculated Q values for the Fc region, Figure 6d shows similar calculated Q values for the PQ region, and Figure 6e shows the calculated values of redox capacity, NOx and NRed. These response values show a linkage between PQ reduction and Fc 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

oxidation: as the potential amplitude is expanded into the range that allows PQ reduction, a response is observed in both PQ reduction and Fc oxidation. (Further analysis of Figure 6 is provided in Figure S4 of Supporting Information.) Confirmatory Studies with Synthetic Cysteinyldopamine-Dopamine Core-Shell Melanin A synthetic model of neuromelanin is the cysteinyldopamine-dopamine core-shell melanin. To test the ability of this synthetic core-shell melanin to undergo reductive redox-cycling with PQ, we repeated the experimental approach of Figure 6 using a chitosan film with this entrapped synthetic model. Figure 7a shows that as the input potential range was progressively increased to allow PQ reduction, then amplified output currents were observed in both the PQ reduction regions and Fc oxidation regions. Figure 7b further illustrates this linked output response by considering CVs from each of these step amplitude increases (3rd cycle). Analogous to the results with Sepia melanin (Figure 6), Figure 7c shows the calculated Q values for the Fc region, Figure 7d shows calculated Q values for the PQ region, and Figure 7e shows the calculated values of redox capacity, NOx and NRed. The response characteristics observed in Figure 7 for the synthetic core-shell melanin are similar to those observed with the Sepia melanin (Figure 6): perturbations in input reducing conditions (that allow PQ reduction) result in perturbations in output oxidative currents associated with Fc oxidation. These results provide additional support for the hypothesis that PQ can redox-cycle with melanin. Response Characteristics of Other Melanin Samples: The Importance of “Tuning” Sepia melanin is primarily a eumelanin containing indole derived structures similar to the surface component of neuromelanin that is also believed to be derived from dopamine.19, 45, 50 Thus, we “tuned” our reverse engineering approach to probe for redox interactions between PQ and dopamine-derived eumelanin. Specifically, we used Fc as the oxidative redox-cycling mediator as illustrated in Figure 8a. Previous studies have shown that synthetic pheomelanin from cysteinyldopa is also redox-active but its redox potential is more oxidative than Fc.39 Figure 8a shows that pheomelanin’s redox potential falls outside the potential range spanned by Fc and PQ, and thus it should not be able to engage in prolonged redox-cycling. In other words, by selecting Fc as our oxidative redox-cycler we have “tuned” our approach to detect redox-cycling with eumelanin but not with pheomelanin. To illustrate this point, we performed the same experiment in Figure 6 with a model synthetic melanin derived from cysteinyldopamine (Figure S5 of Supporting Information) and natural pheoemanin from red hair which contains both cysteinyldopamine and dopamine melanins (Figure S6 of Supporting Information). The results are summarized in Figure 8b which shows the most important signature of PQ redox-cycling (an amplification in Fc oxidation upon PQ is reduction) is absent for the case of the synthetic cysteinyldopamine and weak for the case of human red hair melanin (Figure S7 of Supporting Information shows additional results). This result illustrates the opportunity to tune the reverse engineering approach to probe for specific redox interactions (i.e., PQ-eumelanin redox-cycling). Different tuning (i.e., different mediators) would be required to detect redox-cycling between PQ and cysteinyldopamine.

CONCLUSIONS

8 ACS Paragon Plus Environment

Page 8 of 25

Page 9 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In conclusion, we used a novel electrochemistry-based reverse engineering methodology and observed for the first time that paraquat (PQ) can reductively redox-cycle with melanin. This method isolates PQmelanin interactions by using the electrode as the source of electrons: PQ shuttles electrons to the insoluble melanin through its reductive redox-cycling. The electrode is also the sink of electrons: oxidative redox-cycling with the electrochemical mediator Fc shuttles electrons from the melanin back to the electrode. Cyclic imposed voltages are required to enable the electrode to sequentially serve as the electron source and sink. In essence, this method simplifies the experimental system by using the electrode to replace the biological reductants and oxidants. Importantly, O2 is excluded during these measurements to avoid electron transfer to this oxidant. Previous studies have shown that exposure of reduced melanin films to air-saturated water results in the transfer of electrons to O2 with the generation of reactive oxygen species (ROS).43 We believe the ability to isolate and study specific redox interactions could provide a valuable complement to more traditional experimental approaches in redox-biology. A reverse engineering approach was used previously to demonstrate that natural melanins retain redox activity and can rapidly and repeatedly donate and accept electrons.39 Recent independent observations indicate that melanins can accept electrons from common biological reductants (e.g., NADPH and glutathione)20, 55 and also donate electrons to O2 to generate ROS.43, 56-60 Together, these results suggest that melanins may be an under-appreciated participant in important interactions in redox-biology. Here, we extend these studies to demonstrate that melanin can undergo redox-cycling interactions with a redox-active environmental toxin (PQ). The obvious limitations to reverse engineering studies are that observations of PQ-melanin redox-cycling in vitro do not prove that such mechanisms are operative in vivo, nor do they reveal a role for such a mechanism in disease pathologies. Despite these inherent limitations, we believe the observation that PQ and melanin can interact is important because it demonstrates a redox interaction between two components that have been individually linked to oxidative stress and the etiology of Parkinson’s disease. EXPERIMENTAL SECTION Chemicals. The following were purchased from Sigma-Aldrich: chitosan, 1,1’-ferrocenedimethanol (Fc), paraquat (PQ), catechol, melanin from Sepia officinalis. The water (>18 MΩ) used in this study was obtained from a Super Q water system (Millipore). Chitosan solutions (1%, pH 5.5) were prepared by dissolving chitosan flakes in HCl to achieve a final pH of 5-6. The solutions of catechol and mediator were prepared in phosphate buffer (0.1 M; pH 7.0). Synthetic melanin preparation. 5-S-cysteinyldopamine was prepared on gram scale by the method previously described.61 Identity and purity of the compound was secured by comparison with literature data.62 Cysteinyldopamine melanin was prepared by peroxidase/H2O2 oxidation as previously described.63 The core-shell melanin was prepared by cysteinyldopamine melanin induced oxidation of dopamine (dopamine/cysteinyldopamine melanin 1:1 w/w) as previously described64 with the exception that the oxidation mixtures were not acidified for recovery of the melanin pigments but were washed with water three times before drying by lyophilization. The red hair pheomelanin sample was purified from human hair as previously reported.20 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 25

Preparation of Film-Coated Electrode. For the fabrication of the catechol-modified-chitosan,42 chitosan was electrodeposited on the gold electrode by immersing the gold electrode into a chitosan solution (1%, pH 5.6) and applying a cathodic voltage to achieve a constant current density (6 A/m2, 1 min) using a DC power supply (model 6614C Agilent Technologies) with a two electrode system (Pt foil anode). Next, the chitosan film was modified with catechol by immersing the chitosan-coated electrode into a catechol solution (5 mM, 0.1 M phosphate buffer, pH 7.0) and applying an anodic potential (+0.6 V, 5 min). To prepare for the melanin-chitosan film,43 melanin (2 mg/mL) was suspended in the chitosan solution (0.5 %, pH 5.5) and 20 µL of this suspension was spread onto the standard gold electrode (r = 1 mm). The film was vacuum-dried at 37 °C for 20 min and then immersed in phosphate buffer (pH 7.0; 30 min) to neutralize the chitosan and thus form an insoluble film.

Electrochemical Instruments. Electrochemical measurements (cyclic voltammetry and chronocoulometry) were performed using a three electrode system with Ag/AgCl as a reference electrode and Pt wire as a counter electrode (CHI Instruments 6273C electrochemical analyzer). Most electrochemical experiments were performed in a mixed solution containing both 50 µM Fc and 50 µM PQ in phosphate buffer (0.1 M, pH 7.0). Air was excluded during electrochemical measurements by purging the solution with N2 during the experiment.

ACKNOWLEDGEMENTS The authors gratefully acknowledge financial support from the National Science Foundation (CBET1435957) and the Department of Defense (Defense Threat Reduction Agency; HDTRA1-13-1-0037).

SUPPORTING INFORMATION Additional Information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

FIGURE LEGENDS Scheme 1. (a) Redox-cycling of paraquat (PQ) is believed to contribute to oxidative stress by the depletion of cellular antioxidants and/or the generation of reactive oxygen species (ROS). (b) In vitro electrochemistry-based reverse engineering method to evaluate redox-interaction between insoluble melanin and soluble mediators (i.e., electron shuttles). Figure 1. Initial evaluation of PQ redox-activity. (a) Reverse engineering method uses controlled voltage inputs and two soluble mediators to shuttle electrons between the electrode and insoluble melanin: paraquat (PQ) is hypothesized to reductively redox-cycle to transfer electrons to melanin. Control cyclic 10 ACS Paragon Plus Environment

Page 11 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


voltammograms (CVs) with uncoated gold electrodes show that (b) PQ and (c) Fc have redox potentials that bracket the redox-potential of melanin (a requirement for the reverse engineering approach: all CVs were for 3rd cycle at a scan rate of 2 mV/s). Figure 2. Paired amplification of PQ-reduction and Fc-oxidation is a signature of Sepia melanin’s redoxcycling. Compared with redox-inactive chitosan film (negative control), cyclic voltammogram (CV) of melanin-chitosan film shows the paired amplification of PQ-reduction and Fc-oxidation as observed in catechol-chitosan film (positive control): all CVs were for 3rd cycle at a scan rate of 2 mV/s. Figure 3. Evidence for redox-cycling between PQ and Sepia melanin based on amplified and steady output responses (cyclic potential inputs with Fc). (a) Cyclic voltammograms (CVs) show that melanincontaining films have paired amplifications in PQ reduction and Fc oxidation currents (scan rate: 2 mV/s). (b) Input-output representation illustrates that amplification appears “steady” over 20 cycles. (c) Quantification of output response “signal” in terms of charge transfer (Q) for (d) Fc oxidation/reduction and (e) PQ oxidation/reduction. (f) The redox capacity or “excess” electrons transferred in the presence of melanin for Fc’s oxidation (NOx) and PQ’s reduction (NRed). Figure 4. Non-steady output response in the absence of oxidative redox-cycling (cyclic potential inputs without Fc). (a) Cyclic voltammograms (CVs; scan rate 2mV/s), (b) input-output curves and (c) NRed shows non-steady outputs in the absence of Fc. Figure 5. Evidence that PQ can transfer electrons to Sepia melanin (step potential inputs with both PQ and Fc). (a) Three step input potentials are imposed in the presence of PQ and Fc to probe for melanin’s oxidative discharging and reductive charging. (b) Fc-mediated discharging (oxidation) of the melaninchitosan film. (c) PQ-mediated charging of the melanin-chitosan film. See text for experimental details. Figure 6. Evidence that PQ’s and Fc’s redox-cycling with Sepia melanin are linked (oscillating potential inputs with step increases in amplitude). (a) Input-output curves for film-coated electrodes exposed to cyclic potential inputs with step amplitude increases. (b) CV curves for the different potential input ranges show a signature for redox-cycling (3rd cycle for each potential range): perturbations in input reducing potentials lead to responses in output oxidation currents. Quantification of output response in terms of charge transfer (Q) for (c) Fc oxidation/reduction and (d) PQ oxidation/reduction, and (f) redox capacity for Fc’s oxidation (NOx) and PQ’s reduction (NRed). Figure 7. Evidence that PQ’s and Fc’s redox-cycling are linked for a synthetic cysteinyldopaminedopamine core-shell melanin model. (a) Input-output curves for film-coated electrodes exposed to cyclic potential inputs with step amplitude increases. (b) CV curves for the different potential input ranges show a signature for redox-cycling (3rd cycle for each potential range): perturbations in input reducing potentials lead to responses in output oxidation currents. Quantification of output response in terms of charge transfer (Q) for (c) Fc oxidation/reduction and (d) PQ oxidation/reduction, and (f) redox capacity for Fc’s oxidation (NOx) and PQ’s reduction (NRed). Figure 8. Comparison of response signatures for other melanins. (a) Thermodynamic plot illustrates that the use of the Fc mediator “tunes” the method to detect redox-cycling with dopamine-derived 11 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

eumelanin but not cysteinyldopamine-derived pheomelanin. (b) Signatures for PQ-melanin redoxcycling follow the trend: Sepia melanin > the synthetic core-shell melanin > red human hair melanin > synthetic cysteinyldopamine melanin.

LITERATURE CITED (1) Kamel, F. (2013) Paths from Pesticides to Parkinson's, Science 341, 722-723. (2) Goldman, S. M. (2014) Environmental toxins and Parkinson's disease, Annu. Rev. Pharmacol. Toxicol. 54, 141-164. (3) Franco, R., Li, S. M., Rodriguez-Rocha, H., Burns, M., and Panayiotidis, M. I. (2010) Molecular mechanisms of pesticide-induced neurotoxicity: Relevance to Parkinson's disease, Chem.-Biol. Interact. 188, 289-300. (4) Zhang, J. W., Zhao, Y., Bai, Y. J., Lv, G. C., Wu, J. P., and Chen, Y. (2015) The significance of serum uric acid level in humans with acute paraquat poisoning, Sci. Rep. 5, 5. (5) Rodriguez-Rocha, H., Garcia-Garcia, A., Pickett, C., Li, S. M., Jones, J., Chen, H., Webb, B., Choi, J., Zhou, Y., Zimmerman, M. C., and Franco, R. (2013) Compartmentalized oxidative stress in dopaminergic cell death induced by pesticides and complex I inhibitors: Distinct roles of superoxide anion and superoxide dismutases, Free Radical Biol. Med. 61, 370-383. (6) Baltazar, M. T., Dinis-Oliveira, R. J., de Lourdes Bastos, M., Tsatsakis, A. M., Duarte, J. A., and Carvalho, F. (2014) Pesticides exposure as etiological factors of Parkinson's disease and other neurodegenerative diseases--a mechanistic approach, Toxicol. Lett. 230, 85-103. (7) Drechsel, D. A., and Patel, M. (2009) Differential Contribution of the Mitochondrial Respiratory Chain Complexes to Reactive Oxygen Species Production by Redox Cycling Agents Implicated in Parkinsonism, Toxicol. Sci. 112, 427-434. (8) Kasischke, K. A., Lambert, E. M., Panepento, B., Sun, A., Gelbard, H. A., Burgess, R. W., Foster, T. H., and Nedergaard, M. (2011) Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions, J. Cereb. Blood Flow Metab. 31, 68-81. (9) Leithner, C., and Royl, G. (2014) The oxygen paradox of neurovascular coupling, J. Cereb. Blood Flow Metab. 34, 19-29. (10) Zucca, F. A., Basso, E., Cupaioli, F. A., Ferrari, E., Sulzer, D., Casella, L., and Zecca, L. (2014) Neuromelanin of the Human Substantia Nigra: An Update, Neurotox. Res. 25, 13-23. (11) Gaki, G. S., and Papavassiliou, A. G. (2014) Oxidative Stress-Induced Signaling Pathways Implicated in the Pathogenesis of Parkinson's Disease, Neuromolecular Med. 16, 217-230. (12) Zucca, F. A., Giaveri, G., Gallorini, M., Albertini, A., Toscani, M., Pezzoli, G., Lucius, R., Wilms, H., Sulzer, D., Ito, S., Wakamatsu, K., and Zecca, L. (2004) The Neuromelanin of Human Substantia Nigra: Physiological and Pathogenic Aspects, Pigment Cell Res. 17, 610-617. (13) Segura-Aguilar, J., Paris, I., Muñoz, P., Ferrari, E., Zecca, L., and Zucca, F. A. (2014) Protective and toxic roles of dopamine in Parkinson's disease, J. Neurochem. 129, 898-915. (14) Liu-Smith, F., Poe, C., Farmer, P. J., and Meyskens, F. L. (2015) Amyloids, melanins and oxidative stress in melanomagenesis, Exp. Dermatol. 24, 171-174. (15) Mitra, D., Luo, X., Morgan, A., Wang, J., Hoang, M. P., Lo, J., Guerrero, C. R., Lennerz, J. K., Mihm, M. C., Wargo, J. A., Robinson, K. C., Devi, S. P., Vanover, J. C., D'Orazio, J. A., McMahon, M., Bosenberg, M. W., Haigis, K. M., Haber, D. A., Wang, Y. S., and Fisher, D. E. (2012) An ultraviolet-radiationindependent pathway to melanoma carcinogenesis in the red hair/fair skin background, Nature 491, 449-454.


Page 12 of 25

Page 13 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(16) Premi, S., Wallisch, S., Mano, C. M., Weiner, A. B., Bacchiocchi, A., Wakamatsu, K., Bechara, E. J., Halaban, R., Douki, T., and Brash, D. E. (2015) Photochemistry. Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure, Science 347, 842-847. (17) Munoz-Munoz, J. L., Garcia-Molina, F., Varon, R., Tudela, J., Garcia-Canovas, F., and RodriguezLopez, J. N. (2009) Generation of hydrogen peroxide in the melanin biosynthesis pathway, Biochim. Biophys. Acta 1794, 1017-1029. (18) Zecca, L., Bellei, C., Costi, P., Albertini, A., Monzani, E., Casella, L., Gallorini, M., Bergamaschi, L., Moscatelli, A., Turro, N. J., Eisner, M., Crippa, P. R., Ito, S., Wakamatsu, K., Bush, W. D., Ward, W. C., Simon, J. D., and Zucca, F. A. (2008) New melanic pigments in the human brain that accumulate in aging and block environmental toxic metals, Proc. Natl. Acad. Sci. U.S.A. 105, 17567-17572. (19) Bush, W. D., Garguilo, J., Zucca, F. A., Albertini, A., Zecca, L., Edwards, G. S., Nemanich, R. J., and Simon, J. D. (2006) The surface oxidation potential of human neuromelanin reveals a spherical architecture with a pheomelanin core and a eumelanin surface, Proc. Natl. Acad. Sci. U. S. A. 103, 14785-14789. (20) Panzella, L., Leone, L., Greco, G., Vitiello, G., D'Errico, G., Napolitano, A., and d'Ischia, M. (2014) Red human hair pheomelanin is a potent pro-oxidant mediating UV-independent contributory mechanisms of melanomagenesis, Pigment Cell Res. 27, 244-252. (21) Kim, E., Leverage, W. T., Liu, Y., White, I. M., Bentley, W. E., and Payne, G. F. (2014) Redox-capacitor to connect electrochemistry to redox-biology, Analyst 139, 32-43. (22) Kilmartin, P. A., and Hsu, C. F. (2003) Characterisation of polyphenols in green, oolong, and black teas, and in coffee, using cyclic voltammetry, Food Chem. 82, 501-512. (23) Prehn, R., Gonzalo-Ruiz, J., and Cortina-Puig, M. (2012) Electrochemical Detection of Polyphenolic Compounds in Foods and Beverages, Curr. Anal. Chem. 8, 472-484. (24) Roginsky, V., Barsukova, T., Hsu, C. F., and Kilmartin, P. A. (2003) Chain-breaking antioxidant activity and cyclic voltammetry characterization of polyphenols in a range of green, oolong, and black teas, J. Agric. Food Chem. 51, 5798-5802. (25) Liu, N. A., Liang, Y. Z., Bin, J., Zhang, Z. M., Huang, J. H., Shu, R. X., and Yang, K. (2014) Classification of Green and Black Teas by PCA and SVM Analysis of Cyclic Voltammetric Signals from Metallic OxideModified Electrode, Food Anal. Methods 7, 472-480. (26) Lohmann, W., Hayen, H., and Karst, U. (2008) Covalent Protein Modification by Reactive Drug Metabolites Using Online Electrochemistry/Liquid Chromatography/Mass Spectrometry, Anal. Chem. 80, 9714-9719. (27) van den Brink, F. T. G., Buter, L., Odijk, M., Olthuis, W., Karst, U., and van den Berg, A. (2015) Mass Spectrometric Detection of Short-Lived Drug Metabolites Generated in an Electrochemical Microfluidic Chip, Anal. Chem. 87, 1527-1535. (28) Thorsell, A., Isin, E. M., and Jurva, U. (2014) Use of Electrochemical Oxidation and Model Peptides To Study Nucleophilic Biological Targets of Reactive Metabolites: The Case of Rimonabant, Chem. Res. Toxicol. 27, 1808-1820. (29) Faber, H., Melles, D., Brauckmann, C., Wehe, C. A., Wentker, K., and Karst, U. (2012) Simulation of the oxidative metabolism of diclofenac by electrochemistry/(liquid chromatography/)mass spectrometry, Anal. Bioanal. Chem. 403, 345-354. (30) Pedersen, A. J., Ambach, L., Konig, S., and Weinmann, W. (2014) Electrochemical simulation of Phase I metabolism for 21 drugs using four different working electrodes in an automated screening setup with MS detection, Bioanalysis 6, 2607-2621. (31) Jurva, U., Holmen, A., Gronberg, G., Masimirembwa, C., and Weidolf, L. (2008) Electrochemical generation of electrophilic drug metabolites: Characterization of amodiaquine quinoneimine and cysteinyl conjugates by MS, IR, and NMR, Chem. Res. Toxicol. 21, 928-935.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(32) Lavaggi, M. L., Nieves, M., Cabrera, M., Olea-Azar, C., de Cerain, A. L., Monge, A., Cerecetto, H., and Gonzalez, M. (2010) Structural modifications on the phenazine N,N '-dioxide-scaffold looking for new selective hypoxic cytotoxins, Eur. J. Med. Chem. 45, 5362-5369. (33) Rao, A., Wiley, M., Iyengar, S., Nadeau, D., and Carnevale, J. (2010) Individuals achieve more accurate results with meters that are codeless and employ dynamic electrochemistry, J. Diabetes Sci. Technol. 4, 145-150. (34) Crespo, G. A., and Bakker, E. (2013) Dynamic electrochemistry with ionophore based ion-selective membranes, Rsc Advances 3, 25461-25474. (35) Musholt, P. B., Schipper, C., Thome, N., Ramljak, S., Schmidt, M., Forst, T., and Pfutzner, A. (2011) Dynamic electrochemistry corrects for hematocrit interference on blood glucose determinations with patient self-measurement devices, J. Diabetes Sci. Technol. 5, 1167-1175. (36) Liu, Y., Kim, E., White, I. M., Bentley, W. E., and Payne, G. F. (2014) Information processing through a bio-based redox capacitor: signatures for redox-cycling, Bioelectrochemistry 98, 94-102. (37) Kim, E., Gordonov, T., Liu, Y., Bentley, W. E., and Payne, G. F. (2013) Reverse engineering to suggest biologically relevant redox activities of phenolic materials, ACS Chem. Biol. 8, 716-724. (38) Liu, Y., Kim, E., Lee, M. E., Zhang, B., Elabd, Y. A., Wang, Q., White, I. M., Bentley, W. E., and Payne, G. F. (2014) Enzymatic Writing to Soft Films: Potential to Filter, Store, and Analyze Biologically Relevant Chemical Information, Adv. Funct. Mater. 24, 480-491. (39) Kim, E., Panzella, L., Micillo, R., Bentley, W. E., Napolitano, A., and Payne, G. F. (2015) Reverse Engineering Applied to Red Human Hair Pheomelanin Reveals Redox-Buffering as a Pro-Oxidant Mechanism, Sci. Rep. 5, 18447. (40) Oberacher, H., Pitterl, F., and Chervet, J. P. (2015) "Omics" Applications of Electrochemistry Coupled to Mass Spectrometry - A Review, Lc Gc Europe 28, 138-150. (41) Go, Y. M., and Jones, D. P. (2013) The Redox Proteome, J. Biol. Chem. 288, 26512-26520. (42) Kim, E., Liu, Y., Baker, C. J., Owens, R., Xiao, S., Bentley, W. E., and Payne, G. F. (2011) Redox-Cycling and H2O2-Generation by Fabricated Catecholic Films in the Absence of Enzymes, Biomacromolecules 12, 880-888. (43) Kim, E., Liu, Y., Leverage, W. T., Yin, J. J., White, I. M., Bentley, W. E., and Payne, G. F. (2014) Context-dependent redox properties of natural phenolic materials, Biomacromolecules 15, 16531662. (44) Schroeder, R. L., Double, K. L., and Gerber, J. P. (2015) Using Sepia melanin as a PD model to describe the binding characteristics of neuromelanin – A critical review, J. Chem. Neuroanat. 64–65, 20-32. (45) d'Ischia, M., Wakamatsu, K., Cicoira, F., Di Mauro, E., Garcia-Borron, J. C., Commo, S., Galván, I., Ghanem, G., Kenzo, K., Meredith, P., Pezzella, A., Santato, C., Sarna, T., Simon, J. D., Zecca, L., Zucca, F. A., Napolitano, A., and Ito, S. (2015) Melanins and melanogenesis: from pigment cells to human health and technological applications, Pigment Cell Res. 28, 520-544. (46) Wakamatsu, K., Fujikawa, K., Zucca, F. A., Zecca, L., and Ito, S. (2003) The structure of neuromelanin as studied by chemical degradative methods, J. Neurochem. 86, 1015-1023. (47) Wakamatsu, K., Murase, T., Zucca, F. A., Zecca, L., and Ito, S. (2012) Biosynthetic pathway to neuromelanin and its aging process, Pigment Cell Res. 25, 792-803. (48) Fedorow, H., Tribl, F., Halliday, G., Gerlach, M., Riederer, P., and Double, K. L. (2005) Neuromelanin in human dopamine neurons: comparison with peripheral melanins and relevance to Parkinson's disease, Prog. Neurobiol. 75, 109-124. (49) Liu, Y., Hong, L., Kempf, V. R., Wakamatsu, K., Ito, S., and Simon, J. D. (2004) Ion-Exchange and Adsorption of Fe(III) by Sepia Melanin, Pigment Cell Res. 17, 262-269. (50) Ito, S. (2006) Encapsulation of a reactive core in neuromelanin, Proc. Natl. Acad. Sci. U. S. A. 103, 14647-14648. 14 ACS Paragon Plus Environment

Page 14 of 25

Page 15 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(51) Gagne, R. R., Koval, C. A., and Lisensky, G. C. (1980) Ferrocene as an internal standard for electrochemical measurements, Inorg. Chem. 19, 2854-2855. (52) Bonneh-Barkay, D., Reaney, S. H., Langston, W. J., and Di Monte, D. A. (2005) Redox cycling of the herbicide paraquat in microglial cultures, Mol. Brain Res. 134, 52-56. (53) Kim, E., Liu, Y., Shi, X.-W., Yang, X., Bentley, W. E., and Payne, G. F. (2010) Biomimetic Approach to Confer Redox Activity to Thin Chitosan Films, Adv. Funct. Mater. 20, 2683-2694. (54) Meredith, P., and Sarna, T. (2006) The physical and chemical properties of eumelanin, Pigment Cell Res. 19, 572-594. (55) Napolitano, A., Panzella, L., Monfrecola, G., and d'Ischia, M. (2014) Pheomelanin-induced oxidative stress: bright and dark chemistry bridging red hair phenotype and melanoma, Pigment Cell Res. 27, 721-733. (56) Napolitano, A., Panzella, L., Leone, L., and D'Ischia, M. (2013) Red Hair Benzothiazines and Benzothiazoles: Mutation-Inspired Chemistry in the Quest for Functionality, Acc. Chem. Res. 46, 519528. (57) Sarna, T., and Sealy, R. C. (1984) Free radicals from eumelanins: Quantum yields and wavelength dependence, Arch. Biochem. Biophys. 232, 574-578. (58) Ye, T., Hong, L., Garguilo, J., Pawlak, A., Edwards, G. S., Nemanich, R. J., Sarna, T., and Simon, J. D. (2006) Photoionization thresholds of melanins obtained from free electron laser-photoelectron emission microscopy, femtosecond transient absorption spectroscopy and electron paramagnetic resonance measurements of oxygen photoconsumption, Photochem. Photobiol. 82, 733-737. (59) Jacobson, E. S. (2000) Pathogenic roles for fungal melanins, Clin. Microbiol. Rev. 13, 708-717. (60) Jacobson, E. S., and Hong, J. D. (1997) Redox buffering by melanin and Fe(II) in Cryptococcus neoformans, J. Bacteriol. 179, 5340-5346. (61) Aureli, C., Cassano, T., Masci, A., Francioso, A., Martire, S., Cocciolo, A., Chichiarelli, S., Romano, A., Gaetani, S., Mancini, P., Fontana, M., d'Erme, M., and Mosca, L. (2014) 5-S-cysteinyldopamine neurotoxicity: Influence on the expression of α-synuclein and ERp57 in cellular and animal models of Parkinson's disease, J. Neurosci. Res. 92, 347-358. (62) Zhang, F., and Dryhurst, G. (1994) Effects of L-Cysteine on the Oxidation Chemistry of Dopamine: New Reaction Pathways of Potential Relevance to Idiopathic Parkinson's Disease, J. Med. Chem. 37, 1084-1098. (63) Napolitano, A., De Lucia, M., Panzella, L., and D’Ischia, M. (2008) The “Benzothiazine” Chromophore of Pheomelanins: A Reassessment†, Photochem. Photobiol. 84, 593-599. (64) Greco, G., Panzella, L., Gentile, G., Errico, M. E., Carfagna, C., Napolitano, A., and d'Ischia, M. (2011) A melanin-inspired pro-oxidant system for dopa(mine) polymerization: mimicking the natural casing process, Chem. Commun. 47, 10308-10310.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1


Page 16 of 25

Page 17 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1



Figure 2

Redox Cycling Signature(Sepia Melanin) Reduction PQ2+ + e- PQ+•

3

Current (µA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chitosan (Negative Control) Melanin-Chit Catechol-Chit (Positive Control)

2 1 0 -1 -2 -3

Oxidation Fc Fc+ + e-

0.6

0.3

0

-0.3

-0.6

-0.9

E(V) vs Ag/AgCl


Page 18 of 25

Page 19 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4


Page 20 of 25

Page 21 of 25

Figure 5 Output Response to Step Potential Input(Sepia Melanin) (a) Input for Step Changes in Potential E(V) vs Ag/AgCl

-1

-0.65 V

-0.65 V

Reductive charging

Initial Charging

-0.5 0

+0.5 V

0.5

Oxidative discharging

1 0

30

Time (min)

(b) Output for Oxidative Step

60

90

(c) Output for Reductive Step 0.4

0

Chit -0.2

Charge (mC)

Charge (mC)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Chit QFc ,Ox

Mel-Chit -0.4 -0.6

N Ox =

Mel − Chit Chit Q Fc − QFc ,Ox ,Ox

n ⋅ A⋅ F

= 172 ± 8 nmol/cm 2

-0.8 0

10

20

N Red =

Mel −Chit Chit QPQ, Red − QPQ,Red

n ⋅ A⋅ F = 83 ± 20 nmol/cm 2

0.3 0.2

Mel-Chit

0.1 Chit QPQ, Red

Chit

Mel −Chit QFc ,Ox

Mel −Chit QPQ, Red

0

30

0

10

20

Time (min)

Time (min)


30


Figure 6 Output Response to Perturbations in Cyclic Potential Inputs with Step Increases in Amplitude (Sepia Melanin) Input

(a)

Chit Mel-Chit

Reduction, PQ2+ 0.6

-0.7

Current (µA)

E(V) vs Ag/AgCl

Output 0.9

-1.1

-0.3 0.1

Reduction, PQ2+

0.3 0 -0.3 -0.6

0.5

Oxidation, Fc

Oxidation, Fc

-0.9 0

100

200

300

400

500

600

0

700

100

200

Time (min)

(b)

-0.4 V

0.8

Current (µA)

0.4

EFc

300

400

500

600

700

Time (min)

-0.5 V

0.8

Chit Mel-Chit

-0.6 V

0.8

-0.7 V

0.8

-0.8 V

0.8

Reduction

EPQ

0

-0.4

PQ2+ + e- PQ+•

0.4

0.4

0.4

0

0

0

0

-0.4

-0.4

-0.4

-0.4

0.4

Oxidation Fc Fc+ + e-

0.6 0.3

0

-0.8 -0.3 -0.6 -0.9 0.6 0.3

0

0

-0.3 -0.6 -0.9

E(V) vs Ag/AgCl Charge Transfer for Fc-Redox Reaction Mel-Chit QFc,Red

20

Chit QFc,Red

0 -20 Chit QFc,Ox

-40 -60 -80

Mel-chit QFc,Ox

-100 -0.4

-0.5

-0.6

-0.7

E(V) vs Ag/AgCl

-0.8

0

0.6 0.3

-0.3 -0.6 -0.9

0

-0.3 -0.6 -0.9

Perturbation in Fc-Oxidation Output Responses

(d) 120

40

0.6 0.3

Charge Transfer for PQ-Redox Reaction

Charge (µC)

(c)

-0.8

-0.8

-0.8 0.6 0.3 -0.3 -0.6 -0.9

Redox Capacity

(e)

25

NFilm (nmol/cm2)

-0.8

Charge (µC)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 25

20

Mel-chit

100 80 60 40 20 0 -20 -40

QPQ, Red Chit QPQ,Red

Mel-Chit

Chit

QPQ,Ox

QPQ,Ox

15

NOx

10 5

NRed

0

-0.4

-0.5

-0.6

-0.7

-0.8

E(V) vs Ag/AgCl


-0.4

-0.5

-0.6

-0.7

E(V) vs Ag/AgCl

-0.8

Page 23 of 25

Figure 7 Output Response to Perturbations in Cyclic Potential Inputs with Step Increases in Amplitude (Synthetic Core-Shell Melanin) Input

(a)

Output

Reduction, PQ2+

Chit Mel-Chit

1.3

-0.7

Current (µA)

E(V) vs Ag/AgCl

-1.1

-0.3 0.1

Reduction, PQ2+

0.8 0.3 -0.2

0.5

Oxidation, Fc

Oxidation, Fc

-0.7

0

100

200

300

400

500

600

0

700

100

200

Time (min)

(b)

-0.4 V 1.4

Current (µA)

0.9

EFc

300

400

500

600

700

Time (min)

-0.5 V

-0.6 V

-0.7 V

-0.8 V

1.4

1.4

1.4

0.9

0.9

0.9

0.9

0.4

0.4

0.4

0.4

-0.1

-0.1

-0.1

-0.1

1.4

Chit Mel-Chit

Reduction PQ2+ + e- PQ+•

EPQ

0.4

-0.1

Oxidation 0.6 0.3

0

-0.6 -0.3 -0.6 -0.9 0.6 0.3

0

-0.6 -0.3 -0.6 -0.9 0.6 0.3

-0.3 -0.6 -0.9

E(V) vs Ag/AgCl Charge Transfer for Fc-Redox Reaction

Mel-Chit QFc,Red

20

QChit Fc,Red

0 -20

Chit QFc,Ox

-40 -60

Mel-chit QFc,Ox

-80 -100 -0.4

-0.5

-0.6

-0.7

E(V) vs Ag/AgCl

-0.8

0

100 80 60 40 20 0 -20 -40

0.6 0.3

-0.3 -0.6 -0.9

0

-0.3 -0.6 -0.9

Perturbation in Fc-Oxidation Output Responses

(d) 120

40

0.6 0.3

Charge Transfer for PQ-Redox Reaction

Charge (µC)

(c)

-0.6

-0.6 0

Redox Capacity

(e)

25

NFilm (nmol/cm2)

-0.6

Fc Fc+ + e-

Charge (µC)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20

Mel-chit

QPQ, Red Chit

QPQ,Red

Mel-Chit QPQ,Ox

Chit

QPQ,Ox -0.4

-0.5

-0.6

-0.7

-0.8

E(V) vs Ag/AgCl


15 10

NOx

5 0 -0.4

NRed -0.5

-0.6

-0.7

E(V) vs Ag/AgCl

-0.8


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8


Page 24 of 25

Page 25 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Graphic


ParaquatâMelanin Redox-Cycling: Evidence from Electrochemical

Recommend Documents

ParaquatâMelanin Redox-Cycling: Evidence from Electrochemical