Research Article

Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure

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Science  20 Feb 2015:
Vol. 347, Issue 6224, pp. 842-847
DOI: 10.1126/science.1256022

The dark side of melanin exposed

Sun worshippers may have more to worry about than the DNA damage that occurs while they're relaxing on the beach. It seems that the DNA photoproducts responsible for cancer-causing mutations in skin cells continue to be generated for hours after sunlight exposure. Premi et al. find that a key mediator of this delayed damage is melanin, a pigment thought to protect against cancer (see the Perspective by Taylor). They propose a “chemiexcitation” model in which reactive oxygen and nitrogen species induced by ultraviolet light excite an electron in melanin fragments. This energy is then transferred to DNA, inducing the same damage as ultraviolet light, but in the dark. Conceivably, this energy could be dissipated by adding quenchers to sunscreens.

Science, this issue p. 842; see also p. 824

Abstract

Mutations in sunlight-induced melanoma arise from cyclobutane pyrimidine dimers (CPDs), DNA photoproducts that are typically created picoseconds after an ultraviolet (UV) photon is absorbed at thymine or cytosine. We found that in melanocytes, CPDs are generated for >3 hours after exposure to UVA, a major component of the radiation in sunlight and in tanning beds. These “dark CPDs” constitute the majority of CPDs and include the cytosine-containing CPDs that initiate UV-signature C→T mutations. Dark CPDs arise when UV-induced reactive oxygen and nitrogen species combine to excite an electron in fragments of the pigment melanin. This creates a quantum triplet state that has the energy of a UV photon but induces CPDs by energy transfer to DNA in a radiation-independent manner. Melanin may thus be carcinogenic as well as protective against cancer. These findings also validate the long-standing suggestion that chemically generated excited electronic states are relevant to mammalian biology.

The pigment melanin protects against sunlight-induced burns, DNA damage, and skin cancer. These attributes of melanin are due to an unusually broad absorption spectrum and perhaps radical scavenging activity (1). Yet paradoxes abound. First, melanin is not solely beneficial: Blondes and redheads, who have a higher ratio of yellow pheomelanin to brown eumelanin in their skin and hair, have a greater risk for melanoma than dark-haired individuals (by a factor of 2 to 4), and the pheomelanin/eumelanin ratio accounts for some of this risk (2). UVA-irradiated mice do not develop melanoma if they lack melanin (3), and Braf-mutant mice develop 10 times as many spontaneous melanomas if they carry the pheomelanin-associated Mc1re/e allele (4). UV-irradiated melanin, especially pheomelanin, triggers apoptosis and production of reactive oxygen species (ROS) and DNA strand breaks (5, 6). Melanin synthesis itself generates ROS, especially synthesis of pheomelanin (7).

Yet melanin’s ROS-generating properties do not explain its role in melanoma development, because most mutations in human melanomas display the UV signature of C→T substitutions at sites of adjacent pyrimidines (8, 9). Such mutations arise from cyclobutane pyrimidine dimers (CPDs), which join adjacent pyrimidines to distort the DNA helix (10, 11); genetic disorders of CPD repair, such as xeroderma pigmentosum, elevate the risk for childhood melanoma by four orders of magnitude. The most energetic component of sunlight, UVB (280 to 315 nm), creates these DNA photoproducts almost instantaneously, so the only obvious effect of melanin is shielding. The lower-energy UVA (315 to 400 nm), which constitutes ~95% of the ultraviolet energy that penetrates the atmosphere, also induces melanoma in mice and in humans who use tanning beds, but it is inefficient at making the cytosine-containing CPDs that cause C→T mutations (12, 13). We provide evidence for an unusual biochemical pathway that resolves these oncological discrepancies by making melanin an active participant in CPD formation.

Delayed cyclobutane pyrimidine dimer induction in melanocytes

As a positive control for an unrelated experiment, we exposed murine fibroblasts and melanocytes to radiation from a UVA lamp, using an enzyme-linked immunosorbent assay (ELISA) to measure the number of CPDs over time. Induction of CPDs by direct UV absorption is complete in one picosecond (11). Consistent with this, we found that for fibroblasts and for melanocytes derived from albino mice, the peak of CPD induction was reached immediately upon exposure, followed by slow nucleotide excision repair (Fig. 1A and fig. S1A). (14). Unexpectedly, melanin-containing murine melanocytes instead continued to generate CPD for at least 3 hours after UVA exposure, at which point generation was offset by DNA repair (Fig. 1, B and C). The negative result with albino melanocytes implicated melanin in the process and ruled out the involvement of photosensitizing compounds in the growth medium. We confirmed the delayed production of CPDs by using a comet assay for DNA strand breaks after treating lysed cells with a nucleotide excision repair enzyme to induce DNA breaks at CPD sites (fig. S1B). This result also indicated that delayed CPDs resided in nuclear DNA. Delayed CPDs were also visible by immunofluorescence in normal melanocytes, whereas albino melanocytes showed only repair (fig. S1, C and D). We conclude that pigmented melanocytes induce “dark CPDs” after UV exposure ends.

Fig. 1 Cyclobutane pyrimidine dimers (CPDs) continue to be generated in melanocytes long after UV exposure ends.

(A to C) In albino murine melanocytes (A), CPD induction at time 0 is followed by repair, but in melanin-containing melanocytes [(B) and (C)], CPDs continue to increase for 4 hours after UVA exposure ends. CPDs were assayed by DNA ELISA. (D) Additional dark CPDs are revealed in melanocytes when excision repair is suppressed by siXpa or siXpc; dark CPDs account for half the total CPDs. (E) In melanocytes from humans, the production of dark CPDs after UVA varies between individuals. (F) “Nonproducers” are revealed as producers after DNA repair is suppressed in the cells by siXPA or siXPC. (G) Dark CPDs in mouse melanocytes and keratinocytes in vivo. K14-Kitl transgenic mice, which have epidermal melanocytes containing eumelanin, were crossed to mice carrying the Mc1re/e allele, which confers epidermal pheomelanin and yellow fur. Scale bar, 50 μm. (H) Quantitation of CPDs in epidermal sections. Error bars are SD from four experiments [(A) to (C)], three experiments [(D) to (F)], or two experiments (H). P values by t test are for the difference between the asterisked time point and 0 hours or as indicated. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.00005.

We next examined whether dark CPD induction was masked by concurrent repair and thus might be more extensive than it appeared. Knocking down excision repair of CPDs by means of small interfering RNA (siRNA) against Xpa or Xpc transcripts (corresponding to genes mutated in xeroderma pigmentosum) revealed that dark CPDs constituted half of all CPDs (Fig. 1D). This factor of 2 increase in DNA photoproducts is substantial because cancer-predisposed individuals in xeroderma pigmentosum complementation group D are only 60% defective in CPD repair (15). For the higher-energy UVB, the majority of CPDs in melanocytes were created in the dark, even without repair knockdown (fig. S1, E to H). The UVB process was also dependent on melanin, and thus it differs from the delayed production of CPDs that has been reported occasionally in other cell types after UVC or UVB exposure (1618).

Human melanocytes also generated dark CPDs after UVA or UVB exposure (Fig. 1E and fig. S1I), although there was interindividual variation in the response, particularly for UVA. This likely reflects genetic differences between the donors. For human melanocytes in which there was a modest rate of CPD decline after exposure but which showed no obvious dark CPDs, the dark CPDs were revealed by siRNA knockdown of XPA or XPC transcripts (Fig. 1F). It was not possible to determine whether individual variation was due to variation in melanin type because of privacy restrictions for newborn foreskin tissue.

To examine CPD induction in vivo, we used transgenic mice in which melanocytes remain in the epidermis throughout life because of expression of the Kit ligand in keratinocytes (K14-Kitl), thus mimicking human skin. After exposure of these mice to UVA, the level of epidermal CPDs at the 2-hour time point was 3 times the level immediately after exposure (Fig. 1, G and H). Most epidermal cells were keratinocytes, which receive pigment from the melanocytes; this finding suggests that melanin content, rather than synthesis, was the crucial requirement for dark CPD induction. Furthermore, in K14-Kitl mice homozygous for the inactive Mc1re/e allele, in which melanocytes synthesize red-yellow pheomelanin, both initial and dark CPDs were twice as frequent as in black mice. This suggests that pheomelanin is both a poorer shield against normal CPD formation and a more potent dark CPD generator.

Cytosine-containing dark CPDs

We next used mass spectrometry to identify individual CPD species in murine melanocytes. This analysis revealed that the delayed CPDs in melanin-containing melanocytes included the cytosine-containing CPDs that generate UV-signature C→T mutations (Fig. 2, A and B). Unexpectedly, relative to directly induced CPDs, we observed a higher (TC + CT)/TT CPD ratio (by a factor of 4) in the delayed CPDs, which is suggestive of unusual chemistry. UVA generates primarily TT CPDs, with TC and CT CPDs together contributing only 10 to 30% of these three CPD types (13). The (TC + CT)/TT CPD ratio in melanin-containing cells increased from the UVA-like 0.37 at 0 hours (directly induced CPDs) to 1.33 in the CPDs arising during the next 2 hours (P = 0.027)—a ratio more typical of cells irradiated with the higher-energy UVB. Mass spectrometry ruled out two possibilities: (i) that the ELISA and comet assays cross-reacted with melanin-DNA adducts, and (ii) that the delayed CPD appearance reflected greater access of the antibody or endonuclease due to a change in DNA conformation after UV exposure.

Fig. 2 Mass spectrometry reveals enrichment of dark CPDs for CPDs that contain cytosine.

For energetic reasons, direct absorption of UVA generates primarily thymine-containing (TT) cyclobutane pyrimidine dimers. (A) There is no dark CPD formation in albino melanocytes after UVA exposure. The slope of post-UVA CPD induction in albino cells is indistinguishable from the slope in unirradiated cells. Data represent the average of three experiments, expressed as a fraction of the total CPDs generated at 0 hours. Hence, the total CPD line has no error bar. The total number of CPDs at 0 hours was 169 CPDs per megabase of DNA. (B) Dark CPDs are induced in melanin-containing melanocytes by UVA and include a greater number of cytosine-containing TC and CT dimers, capable of causing UV-signature C→T mutations. Slopes for post-UVA induction of TT, TC, and CT CPDs in melanin-containing cells are greater than those in albino, with P = 0.01, 0.05, and 0.03, respectively. The total number of CPDs at 0 hours was 87 CPDs per megabase of DNA, consistent with the shielding function of eumelanin. Data represent the average of five experiments.

Photochemical pathway

To identify the photochemical pathway that produces dark CPDs, we focused on UVA because it reduces background from CPDs created by direct UVB absorption, reduces photosensitization from aromatic molecules, and is used in indoor tanning beds. We found that kojic acid—an inhibitor of tyrosinase, the rate-limiting enzyme in melanin synthesis—suppressed production of dark CPDs by 85% (Fig. 3A). Because ROS can be produced in cells for long periods after UVA exposure (19), we tested the ROS scavengers N-acetylcysteine and α-tocopherol (vitamin E). The former suppressed production of dark CPDs by 64% and the latter abolished it (fig. S2, A and B). An effect of antioxidants on CPDs detectable immediately after UV has been reported, but was linked to changes in chromatin structure (20). Because melanin generates superoxide radical ion (O2•–) while it is being irradiated, we specifically scavenged it with TEMPOL (1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine) and found that this also abolished the production of dark CPDs after UVA (fig. S2C). A longer-lasting source of O2•– in cells is NOX [reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase], which is rapidly induced by UVA (19). The NOX inhibitor VAS2870 also abolished production of dark CPDs (Fig. 3B). CPDs were inhibited to a level below that at 0 hours, indicating that dark CPDs were also induced during the irradiation itself.

Fig. 3 In the proposed photochemical pathway for production of dark CPDs, high-energy triplet-state moieties are created by melanin, superoxide, and nitric oxide.

Dark CPDs are blocked by inhibitors of (A) melanin synthesis (kojic acid, KA), (B) the superoxide generator NADPH oxidase (VAS2870), and (C) the nitric oxide generator iNOS (aminoguanidine). Inhibitors are not expected to reduce CPD levels below those seen at 0 hours, a time when CPDs are generated primarily by direct UV absorption (dotted line). (D) The product of superoxide and nitric oxide, peroxynitrite, generates 3-nitrotyrosine–modified proteins in melanocyte nuclei (except in albino melanocytes), and these increase with UVA exposure. (E) Quantitation of 3-nitrotyrosine in melanin-containing melanocytes. The increase at 0 hours arises from protein nitration during the 27-min UVA exposure. (F) Ultraweak chemiluminescence is generated by permeabilized UVA-irradiated melanized melanocytes that had been incubated with a triplet energy acceptor probe, DBAS. Chemiluminescence was quantified by single-photon counting (cpm, counts per minute). (G) Albino melanocytes do not produce chemiluminescence when irradiated. (H) A triplet-state quencher, ethyl sorbate, inhibits production of dark CPDs in pigmented mouse melanocytes after they are exposed to UVA. Data are the average of three experiments. (F) and (G) show data from one of three similar experiments. P values are for the difference between treated and untreated samples.

The O2•– requirement recalled an old observation (21) that CPDs can be generated in the dark by dioxetane, a strained four-member ring peroxide. This moiety undergoes thermolysis to two carbonyls, with one in an electronically excited triplet state having the high energy of a UV photon (>3 eV, 70 kcal/mol, or 35,000 K) and capable of transferring its energy to DNA by a process that is independent of radiation (21). One of the few biologically synthesized molecules that can initiate such “photochemistry in the dark” (22, 23) is peroxynitrite (ONOO) (24), which is generated by the reaction of O2•– with nitric oxide (NO). Peroxynitrite and its precursors are stable enough to diffuse several cell diameters from the site of generation to the target molecule. NOX and the NO generator iNOS are UV-inducible within minutes and are expressed in melanocytes, including nuclear NOX4 (25). To determine whether NO is required for production of dark CPDs, we inhibited iNOS by treating melanocytes with aminoguanidine. This resulted in complete suppression of dark CPD production (Fig. 3C).

We next investigated whether melanin-containing cells generate peroxynitrite when exposed to UV. Peroxynitrite can be detected by its nitration of tyrosines. Assays for 3-nitrotyrosine revealed that basal nitration activity was present even without UV, that it was located primarily in the nucleus, and that its appearance required melanin (Fig. 3, D and E, and fig. S2D). The melanin requirement suggests that the NO requirement in melanocytes is distinct from that observed in cultured keratinocytes (18). Peroxynitrite’s presence in the nucleus without UV exposure is plausible because the synthesis of melanin monomers is a redox reaction that releases O2•– (7) and melanosomes are assembled in the perinuclear space. In support of an additional UV role, we found that exposure of melanin-containing melanocytes to UVA created as much nuclear and cytoplasmic nitrotyrosine in 30 min as had accumulated from basal induction over ~8 days (Fig. 3, D and E, and fig. S2D) (14). This represents a factor of ~400 spike in the flux of peroxynitrite per hour.

A diagnostic for excited triplet states is that their energy can also discharge via ultraweak blue or green luminescence (23). This signal can be enhanced by several orders of magnitude by diverting the energy to a triplet energy acceptor, sodium 9,10-dibromoanthracene-2-sulfonate (DBAS), which is a more efficient emitter. To allow rapid DBAS entry, we permeabilized melanocytes within a liquid scintillation counter set to single-photon counting mode. UVA-irradiated melanocytes generated DBAS-amplified luminescence for several hours after irradiation, and only in cells containing melanin (Fig. 3, F and G). The lifetime of triplet carbonyls is ~10 μs (24), so a signal persistent over the course of hours indicates ongoing creation. To test whether this triplet state is required for dark CPDs, we added ethyl sorbate, a specific quencher of triplet states (26), to intact melanocytes after UVA exposure. We found that this treatment prevented production of dark CPDs (Fig. 3H). Because DBAS diverts triplet energy to luminescence, this compound also blocked dark CPDs (fig. S2E). These results provide direct evidence that UV-induced superoxide and nitric oxide lead to a high-energy triplet state moiety, which then creates a dark CPD by energy transfer.

Electronically excited melanin as a molecular vector

Melanin is located in the cytoplasm, yet CPDs were generated in the nucleus, raising the question of what molecule acquired the energetic carbonyl. An O2•–-initiated radical chain reaction in lipids was one possible conduit (26), but inducing lipid peroxidation in isolated nuclei by exposure to cumene hydroperoxide did not appear to induce CPDs in nuclear DNA (fig. S3).

Another candidate for the triplet-state carrier was the set of melanin monomers out of which the final melanin polymers are assembled. These monomers are lipophilic and are therefore potentially able to enter the nucleus, and are concentrated in perinuclear coated vesicles before transfer to melanosomes (27). Melanin polymer rapidly solubilizes when exposed to hydrogen peroxide (H2O2) (28), and its degradation by peroxidation or UV photoionization has been proposed to involve dioxetane and triplet carbonyls (29, 30). We first investigated whether a triplet state could be created in melanin by a cell-free system. Synthetic melanin oxidized with peroxynitrite and incubated with DBAS generated a chemiluminescence signal two orders of magnitude larger than that seen in melanocytes (Fig. 4A and fig. S4A). We found that chemiluminescence began immediately and proceeded for >10 min. To further test melanin’s ability to host a triplet state, we oxidized synthetic melanin with horseradish peroxidase in the presence of hydrogen peroxide, which generates dioxetane intermediates by a mechanism similar to that of peroxynitrite (24). This produced similar levels of chemiluminescence (Fig. 4A and fig. S4B). The eumelanin monomer 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and the pheomelanin monomer 5-S-cysteinyldopa (5SCD) also generated chemiluminescence when oxidized (Fig. 4A).

Fig. 4 Excited-state melanin may act as a molecular vector.

(A) Synthetic melanin, its eumelanin monomer DHICA, and its pheomelanin monomer 5SCD generate ultraweak chemiluminescence when oxidized by peroxynitrite or by horseradish peroxidase plus H2O2. DBAS was used as a triplet energy probe, so luminescence indicates the presence of electronically excited triplet states. Data represent one of six experiments. (B) CPDs are created in the dark when melanin or its monomers are oxidized with peroxynitrite. The level of CPDs resulting from direct DNA absorption of UVA is shown for comparison. (C) Diverting triplet energy to luminescence with DBAS blocks production of CPDs by oxidized melanin. The UVC dose used for comparison is about two orders of magnitude higher than that lethal to 50% of normal cells. In (B) and (C), data represent the average of three experiments. P values are for the difference between treated and “DNA only” samples or as indicated. (D) Synthetic melanin is rapidly solubilized upon exposure to UV or peroxynitrite. Left to right: Supernatants resulting from untreated melanin or from melanin exposed to UVA (200 kJ/m2), 7.8 mM NaOH, or peroxynitrite (1 mM in NaOH stabilizer). (E and F) Identification of a putative triplet-carbonyl carrier. (E) The eumelanin monomer DHICA was oxidized and products were separated by HPLC, monitoring the HPLC output by mass spectrometry in negative ion mode. Monitoring for the 224 m/z ion of 225-dalton compounds 1 and 2, expected for triplet-carbonyl derivatives of dioxetane-adducted DHICA, reveals a single polar peak. (F) Scanning the polar HPLC peak reveals four principal m/z peaks, one at 224 m/z.

We found that CPDs were generated in the complete absence of UV when we incubated plasmid DNA and peroxynitrite with melanin, DHICA, or 5SCD (Fig. 4B). We were unable to test whether the triplet quencher ethyl sorbate blocked these reactions because it was insufficiently soluble in the aqueous buffer. However, we found that DBAS, which redirects triplet energy toward luminescence, reduced CPD production by 50 to 90% (Fig. 4C and fig. S4C). The CPDs created by oxidizing melanin or its monomers included the mutagenic cytosine-containing CPDs (fig. S4D). The level of CPDs induced in the absence of UV was approximately equal to that generated in pure DNA by 25 kJ/m2 of UVA—an exposure about one-quarter of that required to produce a barely perceptible sunburn (the “minimal erythema dose”). On the basis of our mass spectrometry data from albino murine melanocytes (Fig. 2), this value is approximately 1 CPD per 24 kb of DNA created solely by oxidized melanin.

We next explored whether UV exposure overcomes the migration barrier posed by the nuclear membrane. Cell-free experiments revealed that melanin polymer was rapidly solubilized after exposure to UVA or peroxynitrite (Fig. 4D). Because antibodies to melanin monomers do not exist, we were unable to directly monitor melanin migration into the nucleus. We therefore imaged unfixed melanocytes (to avoid artifactually permeabilizing the nuclear membrane), using differential interference contrast microscopy to maintain contrast at high magnification. Unirradiated normal melanocytes, but not albino melanocytes, showed dark granules in the cytoplasm, especially in the perinuclear area (fig. S5). We presume that these are melanin aggregates in melanosomes, coated vesicles, and endoplasmic reticulum because they have the same size and cellular locations as granules seen in melanocytes immunostained for tyrosinase (31). After UVA exposure, 3D images reconstructed from serial planar images revealed granules inside the nucleus (movies S1 and S2). We do not know whether these represent melanin-containing organelles that moved into the nucleus or molecular aggregates formed by spontaneous polymerization of melanin precursors, as occurs during normal melanin synthesis. We conclude that in cells exposed to UV, melanin or its constituents are able to enter the nucleus.

Finally, we sought to identify carbonyl-containing DHICA reaction products remaining after the triplet excitation energy discharges. The milder reaction of horseradish peroxidase with hydrogen peroxide was used to avoid complete oxidation and thereby reveal labile reaction intermediates. The DHICA analog of the predicted eumelanin degradation product (29) (Fig. 4E, structure 1) has a molecular weight of 225 daltons and would be highly polar. An alternative reaction is formation of a dioxetane moiety at the pyrrole ring (32), also leading to a polar structure of 225 daltons (structure 2). When we separated reaction products by high-performance liquid chromatography (HPLC) and scanned the HPLC eluate by mass spectrometry in negative ion mode for the corresponding 224 m/z product, we observed a single peak eluting in the highly polar region of the buffer gradient (Fig. 4E). Scanning this fraction of the HPLC eluate across the small-molecule region of the mass spectrometer revealed four principal m/z peaks, of which the second largest was 224 daltons (Fig. 4F). Fragmentation of these HPLC fractions generated m/z peaks assignable to oxidation products of either structure 1 or 2 created by known oxidation reactions. When we blocked the reactivity of DHICA’s six-membered ring by omitting an OH group (5-hydroxyindole-2-carboxylic acid) (fig. S6A), oxidation still produced chemiluminescence (fig. S6, B and C) and CPDs (fig. S6D), which suggests that 2 is the predominant product. Together, these results indicate that melanin and melanin fragments are capable of acting as the molecular vectors that acquire an electronically excited triplet state, probably at a carbonyl arising from a dioxetane intermediate.

Proposed scheme for the participation of melanin in melanoma development

We have shown that exposing melanin-containing cells to UV radiation induces superoxide and nitric oxide, causing a factor of ~400 peroxynitrite spike that degrades melanin, allows melanin-like granules to appear in the nucleus and, for hours after the original UV exposure, excites melanin derivatives to a triplet state that has the high energy of a UV photon (Fig. 5). These evanescent electronically excited products transfer their triplet energy to DNA, creating mutagenic cyclobutane pyrimidine dimers in the dark. We speculate that the degradation of melanin polymer into fragments allows these moieties to closely approach the DNA, and that the peroxynitrite-melanin reaction intermediate is a short-lived dioxetane (fig. S6E) whose triplet energy level lies well above that of the DNA bases. The sustained time course of dark CPD generation can be accounted for by the prolonged steps of UV induction of NOX and iNOS, peroxynitrite-induced solubilization of melanin to fragments (or release of pre-melanin monomers from UV-damaged melanosomes and vesicles), and migration to the nucleus. Still unidentified are the isozymes generating superoxide and nitric oxide, the cell site(s) at which melanin is degraded and its fragments are excited to a triplet state, the full inventory of eumelanin and pheomelanin fragments that can host triplet states, the chemical intermediate through which ONOO creates the triplet carbonyl, and the energy transfer process.

Fig. 5 A mechanistic model for the generation of dark CPD in melanocytes by chemiexcitation, with melanin as an active participant.

Exposing cells to UV radiation is known to up-regulate iNOS, NADPH oxidase (NOX), and enzymes of melanin synthesis, presumably causing sustained generation of nitric oxide (NO) and superoxide (O2•–). Cytoplasmic NOS and NOX are indicated on the figure but some isoforms are located in the nucleus. The present experiments show a UV-induced surge in the product of these two radicals, the powerful oxidant peroxynitrite (ONOO), and show that peroxynitrite degrades melanin polymer to fragments. Melanin or melanin fragments then appear in the nucleus. Peroxynitrite is also one of the few biologically synthesized molecules capable of exciting an electron to a triplet state. The present experiments show that on a faster time scale, peroxynitrite excites an electron in a melanin fragment to a triplet state that has the high energy of a UV photon. The typical triplet-state reaction intermediate, not demonstrated here (hence indicated in italics), is a cycloaddition of –O–O– to create an unstable dioxetane; dioxetanes undergo spontaneous thermolysis to yield two carbonyls, one of which acquires most of the energy and finishes in a high-energy triplet state (*). For the melanin-related triplet, the half-life of the reaction intermediate appeared to be minutes, and a carbonyl consistent with a dioxetane precursor was identified by mass spectrometry. Triplet energy then discharges on a microsecond time scale to generate visible luminescence, or discharges in a radiation-independent manner to DBAS (to be emitted as fluorescence), to sorbate (to be dissipated as isomerization and heat), or evidently to DNA bases (where it makes CPDs). The presence of melanin, activation of iNOS and NOX, and the triplet state were shown to be required for dark CPD formation.

A consequence of these events is that melanin may be carcinogenic as well as protective against cancer. This double nature would explain the apparent cancer-facilitating effects of melanin seen in mice and in human epidemiology (24, 6). Melanin is an unusual polymer whose properties set the stage for the events we have described (33). Highly reactive o-quinones created by ROS-generating redox transformations of tyrosine polymerize spontaneously into oligomers. The o-quinones readily accept an electron to become semiquinone radicals, giving melanin a high concentration of free radicals in stable redox equilibrium and stabilized by metal ions. This macromolecule is a photon trap that also acts as an electron-proton photoconductor. These characteristics give melanin its broad light absorption, radical scavenging, and metal reservoir properties, but at a price. First, melanin synthesis generates O2•– and H2O2. UV exposure additionally excites the rings to an energy that, especially for pheomelanin, ejects an electron that is captured by oxygen to yield more O2•– (5, 34). Second, the reactive semiquinones allow melanin to be degraded and these fragments to be adducted to create high-energy unstable moieties such as dioxetanes. Although most of the cell’s melanin synthesis is safely isolated inside melanosomes, the early steps occur in close proximity to the nucleus.

It was proposed long ago that chemiexcitation—the creation of chemical reaction products containing excited electrons that underlie bioluminescence in lower organisms—has broad importance in biology (22, 23). Our data suggest that this may be the case in human skin. The consequence is that half or more of the CPDs in a melanocyte arise after UV exposure ends. In vivo the same appears to be true of keratinocytes, which receive melanosomes donated by melanocytes. If the same holds for human skin, this would mean that past measurements of CPDs immediately after UV exposure have underestimated the consequences of UV exposure.

One benefit of dark photochemistry’s slow course is that it allows intervention. A blocker of dark CPDs, α-tocopherol (vitamin E), is not only an antioxidant but also inactivates dioxetanes by converting them to a pair of diols (35). The triplet quencher ethyl sorbate is an analog of the widely used food preservative potassium sorbate. Screening for novel triplet quenchers offers the prospect of developing “evening-after” sunscreens that could potentially prevent the carcinogenic processes occurring in the skin hours after sunlight exposure ends.

Supplementary Materials

www.sciencemag.org/content/347/6224/842/suppl/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S6

Movies S1 and S2

References (3639)

References and Notes

  1. See supplementary materials on Science Online.
  2. Acknowledgments: We thank M. Bosenberg and V. Muthusamy for UVA-irradiated mouse skin; the Yale Office of Environmental Health and Safety for the single-photon liquid scintillation counter; A. Bommakanti for photography; and D. Mitchell and A. Mennone for helpful discussions. Supported by Department of Defense CDMRP grants CA093473P1 and CA093473 (D.E.B. and R.H.); NIH grant 2 P50 CA121974 (R.H. and D.E.B.); a postgraduate fellowship from the University of Veterinary Medicine, Vienna (S.W.); Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) doctoral fellowship 09/02062-8 (C.M.M.); and FAPESP grant 06/56530-4 and the INCT–Processos Redox em Biomedicina (E.J.H.B.). Confocal support was provided by NIH grant P30 DK34989 to the Yale Liver Center.
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