Reverse Engineering Applied to Red Human Hair Pheomelanin Reveals Redox-Buffering as a Pro-Oxidant Mechanism

Abstract

Pheomelanin has been implicated in the increased susceptibility to UV-induced melanoma for people with light skin and red hair. Recent studies identified a UV-independent pathway to melanoma carcinogenesis and implicated pheomelanin's pro-oxidant properties that act through the generation of reactive oxygen species and/or the depletion of cellular antioxidants. Here, we applied an electrochemically-based reverse engineering methodology to compare the redox properties of human hair pheomelanin with model synthetic pigments and natural eumelanin. This methodology exposes the insoluble melanin samples to complex potential (voltage) inputs and measures output response characteristics to assess redox activities. The results demonstrate that both eumelanin and pheomelanin are redox-active, they can rapidly (sec-min) and repeatedly redox-cycle between oxidized and reduced states, and pheomelanin possesses a more oxidative redox potential. This study suggests that pheomelanin's redox-based pro-oxidant activity may contribute to sustaining a chronic oxidative stress condition through a redox-buffering mechanism.

Figure 1

(a) Putative structures of eumelanin and pheomelanin. (b) Electrochemical approach to reverse engineer melanin: (i) insoluble melanin sample is entrapped in non-conducting film adjacent to electrode; (ii) soluble mediators shuttle electrons between electrode and sample; (iii) complex input potentials (voltages) are applied; and (iv) measured output currents are analyzed.

Figure 2. Raman characterization of synthetic melanin samples and melanin-chitosan films.

(a) Synthetic eumelanin. (b) Synthetic pheomelanin.

Figure 3. Initial electrochemical characterization of synthetic melanins.

Cyclic voltammograms of films containing (a) synthetic eumelanin samples and (b) synthetic pheomelanin samples compared with the control chitosan films (scan rate of 2 mV/s) in the presence of 3 mediators. (c,d) Thermodynamic plots illustrating electron exchange between the synthetic melanins and the 3 mediators for reductive redox-cycling reaction. The D region is unique to pheomelanin. (e,f) Thermodynamic plots for oxidative-redox cycling reaction between synthetic melanins and 3 mediators.

Figure 4. Probing redox potential regions for signatures of redox-cycling (2 mediators).

Cyclic voltammograms (CVs) for (a,c,e) synthetic pheomelanin-chitosan films, and (b,d,f) synthetic eumelanin-chitosan films (scan rate of 2 mV/s). Pheomelanin shows redox activity at a more oxidative potential compared to eumelanin.

Figure 5. Repeated probing of the oxidative potential window for evidence of synthetic pheomelanin’s reversible redox-activity.

(a) Sequence of input voltages used to probe the pheomelanin’s oxidative potential window. Current output for pheomelanin-chitosan film expressed as (b) cyclic voltammogram or (c) output curve. Current output for eumelanin-chitosan film expressed as (d) cyclic voltammogram or (e) output curve. Pheomelanin’s paired amplification of oxidation-reduction currents and the “steady” output indicates pheomelanin’s oxidative redox activity is reversible.

Figure 6. Dynamic probing of the synthetic pheomelanin’s oxidative potential window at varying scan rates.

(a) Schematic illustrating that scan rate affects the depth of film being probed. (b) Parameter ratios used for characterizing redox cycling in the pheomelanin’s oxidative potential window (illustrative CV scan for pheomleanin-chitosan film; 2 mV/s). (c) Amplification ratio shows the greatest difference from control chitosan film is observed at the low scan rates (as expected). (d) Rectification ratios shows that Fc serves as an oxidant with eumelanin (RR_Fc < 1) but a reductant for pheomelanin (RR_Fc > 1).

Figure 7. Imposing step potential changes to probe charge transfer.

(a) Sequence of step changes allows probing for oxidation and reduction. (b) Charge transfer observed after a step change for oxidation (Q_Film and N_Film are quantitative measures of electron transfer). (c) Charge transfer observed after step change for reduction. (d) Summary of N_Film values for various oxidation-reduction steps with synthetic melanins.

Figure 8. Evidence that natural melanins are redox-active.

Cyclic voltammograms of films containing melanin samples (scan rate of 2 mV/s) in the presence of 3 mediators. The paired amplification in oxidation and reduction currents provides evidence that the natural melanins are redox-active and can undergo both oxidative and reductive redox-cycling.

Figure 9. Evidence that natural pheomelanin has a greater redox-based pro-oxidant activity.

Three different input potentials were imposed on samples (upper panels), data were analyzed as a ratio of reductive to oxidative charge transfer (middle panels), and results with natural melanin were compared to results for mixtures of synthetic eumelanin and synthetic pheomelanin. (a) Cyclic voltammetry. (b) Chronocoulometry. (c) Cyclic voltammetry for multiple cycles.