Effect of UV on the susceptibility of acid-soluble Skh-1 hairless mouse collagen to collagenase.

BACKGROUND/PURPOSE: Photoaging of the skin is a result of chronic exposure to environmental ultraviolet radiation (UV). The milieu provided by the extracellular matrix, which significantly influences the behaviour of resident fibroblasts, depends critically on the supermolecular collagen structure. We ask whether direct photochemical treatment of type I collagen with solar wavelengths capable of reaching the dermis can modify the substrate's susceptibility to collagenase in a model in vitro system. METHODS: Acid- extracted Skh-1 hairless mouse collagen samples were irradiated with 0-140 J/cm2 of radiation from bank of filtered FS lamp (UVB/UVA = 0.33, fluence rate = 0.81 mW/cm2). Subsequent to UV irradiation, collagen samples were coupled with fluorescein isothiocyanate (FITC) and assayed for susceptibility to bacterial collagenase by monitoring the appearance of supernatant FITC fluorescence (a measure of lysed collagen) over time of incubation. As a 'reference', unirradiated commercial FITC-labelled citrate-soluble collagen (Elastin Products, Owensville, MO 65066, USA) was similarly analysed. RESULTS: Unirradiated mouse collagen had a lower rate of cleavage than did the calfskin sample. Irradiation of unlabelled mouse collagen for 0-48 h (0-140 J/cm2 total UV) rendered the sample more soluble, with concomitant chain degradation, cross-linking and loss of intrinsic collagen fluorescence. At irradiation time's >/= 4 h (>/=11.7 J/cm2), the irradiated collagen was significantly more susceptible to bacterial collagenase digestion. DISCUSSION: It appears that the rate of cleavage depends on the superstructure of the collagen, since the kinetics of collagen cleavage differ for two collagen samples having essentially the same primary structure. Cleavage kinetics may depend on the 'maturity' (solubility) of the collagen. The observation that UV-damaged mouse collagen is a better substrate for collagenase than the intact sample may be illustrative of a mechanism whereby damaged collagen targets itself for selective attack by collagenase.

Effect of UV irradiation on type I collagen fibril formation in neutral collagen solutions.

BACKGROUND: Collagens have the well-known ability to spontaneously self-associate to form fibrils at physiological temperature and neutral pH in vitro and in vivo. Because solar UV may photochemically alter collagen, the kinetics of fibril formation may be modified. Thus, we have begun a systematic study of the effect of various UV wavebands on fibril formation. METHODS: Citrate-soluble calf skin collagen (Elastin Products) was dissolved at 0.05% in 0.5 M HOAc, dialyzed over 2 days into two changes of 0.0327 M phosphate buffer, pH 7.0 at 4 degrees C, and centrifuged at 48,000 x g. Photolysis was carried out at 4 degrees C with either (a) UVC (UVG-11 lamp), (b) filtered solar-simulating radiation (SSR) or UVA (SSR or UVL-21 lamp filtered with a 2.0 mm Schott WG 345 filter). Gelation was commenced by rapidly raising the temperature from 8 degrees C to 33 degrees C. Nucleation and growth were followed by turbidimetric measurements at 400 nm. RESULTS: UVC radiation (0-17.3 J/cm2) resulted in a dose-dependent decrease in the rate of fibril growth. Under these conditions, concomitant collagen crosslinking and degradation occurred. Fibril nucleation, a prerequisite for growth, was rapid (threshold approximately 2 min) and was not affected by UVC, UVA or SSR. SSR (0-1,320 J/cm2) caused a small decrease in growth rate and in the degree of fibril formation. UVA radiation (0-1,080 J/cm2) had a similar effect. "Direct" photochemical damage thus paralleled absorption via various collagen chromophores, with UVC>SSR approximately UVA. The presence of riboflavin (RF) resulted in groundstate interactions that markedly altered both nucleation and growth kinetics. Irradiation with 29.6 J/ cm2 UVA in the presence of RF photosensitizer caused relatively minor additional changes in fibrillation kinetics. CONCLUSIONS: These results collectively indicate that fibril formation is markedly dependent on specific ground state interactions and relatively insensitive to nonspecific UV damage. On the other hand, fibrils thus formed from photochemically altered collagen may have altered structural properties that could have subtle but unfavorable effects on the local dermal milieu in vivo. Notwithstanding, the relative insensitivity of fibrillogenesis to non-specific photochemical damage probably represents a favorable adaptation, overall, which tends to conserve the mechanical integrity of the skin.

Protection against photodynamic therapy (PDT)-induced photosensitivity by fabric materials.

"Special" highly protective fabrics are now available that offer broad-spectrum protection in preventing sunburn, and possibly other types of photodamage. It is important to know to what extent these fabrics are capable of protecting the wearer against skin cancer, photosensitivity disorders, and inadvertent phototoxic reactions from photodynamic therapy (PDT). We assess the ability of one such special (Solumbra) fabric and one "typical" summer fabric to provide protection against PDT phototoxicity produced in tape-stripped Sk-1 hairless mice by topical 5-aminolevulinic acid (ALA) and (primarily) visible light (360-800 nm). Since ALA-derived photosensitizers absorb most of the visible spectrum, results from these studies give a good indication of the photoprotective capability of these fabrics throughout this region. Mice were irradiated dorsally with a Kodak slide projector equipped with a 300 W tungsten-halogen lamp (I0 = 48.3 mW/cm2). After determining the minimal phototoxic dose (MPD) to be 1.40 +/- 0.4 J/cm2, we irradiated the tape-stripped ALA-sensitized mice through the stretched test fabrics with appropriate multiples of the MPD. The special fabric provided protection against 25-30 MPD visible light between 360-800 nm in 14/14 mice. The typical fabric failed to provide protection against 2.5 MPD of the same spectrum. No phototoxic or other adverse responses were seen with either the ALA or light control. In conclusion, the Solumbra fabric is much more protective against ALA photosensitization than the typical fabric. Both appear better at blocking UV than visible light.

Photoprotection of mammalian acid-soluble collagen by cuttlefish sepia melanin in vitro.

Several important clinical conditions can result in close association between the pigment melanin and dermal collagen. Because melanin and its precursors can be chemically reactive in ground and excited states, it is important to know whether the resulting melanin-collagen interaction results in photoprotection or photoaggression. Acidic and neutral air-saturated collagen suspensions (0.033%) were irradiated with 0-2.6 x 10(4) J/m2 UVC or with 0-83 x 10(4) J/m2 solar-simulating UV radiation (SSR). Photochemical destruction of a photolabile collagen fluorophore (lambda em 360 nm) and collagen chain degradation were monitored as functions of irradiation time in the presence and absence of added (0-100 micrograms) sepia eumelanin. Melanin retarded collagen photodamage but did not qualitatively alter the fluorescence fading kinetics. Both H2O2 and O2-. can be produced by UV irradiation of eumelanin. Added H2O2 and KO2 destroyed collagen fluorescence and caused 50% chain degradation at ca 10-20-fold molar excess. Previous studies have demonstrated that eumelanins efficiently scavenge O2-.. We demonstrated that eumelanin also efficiently scavenges H2O2 as evidenced by its ability to (a) compete with scopoletin for peroxide uptake and (b) directly take up H2O2 through a dialysis bag. The latter observation suggests that peroxide scavenging could occur in vivo by melanin sequestered in melanophages. Thus, neither UV-generated O2-. nor H2O2 are likely to be present in concentrations high enough to cause measurable collagen damage. Absorption and/or scattering of excitation radiation away from the target chromophore appears to be the primary photoprotection mechanism, although scavenging of active O2 intermediates may play an important, if subtle role.

Electron transfer and photoprotective properties of melanins in solution.

The polyquinoid nature of eumelanin(s) enables them to couple oxidation of electron donors with the reduction of electron acceptors. We have studied the ability of synthetic (Sigma) and "biological" (cuttlefish sepia) melanins to mediate electron transfer between hydroxybenzene donors (tyrosine, dopa, chemical depigmenters) and model acceptors (ferricyanide, tyrosinase). 1) Depending on the reductant, melanin either retards or accelerates ferricyanide reduction. Reaction kinetics are consistent with a mechanism involving non-interactive binding of both hydroxybenzene and ferricyanide to melanin prior to coupled electron transfer. 2) Melanins also act as an electron conduit in markedly accelerating the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH). The active species appears to be a complex between melanin and MMEH. The magnitude of both effects depend on the type of melanin as well as its oxidation state. Sepia (eu)melanin appears to protect against UV-induced damage to acid-soluble collagen, as judged by irreversible loss of intrinsic collagen fluorescence. Photoprotection against this type of damage appears primarily to involve optical absorption/scattering by the pigment.

Chronic exposure of Sk-1 hairless mice to narrow-band ultraviolet A (320-355 nm)

Several recent investigations collectively suggest that the role of ultraviolet A (UVA) in chronic actinic skin damage may be greater than originally thought. In the present work, the output of a xenon-arc solar-simulator passed through a Bausch & Lomb monochromator in conjunction with a 2-mm Schott WG-320 filter produced narrow-band UVA centered at 338 nm, half-band width 24 nm, I0 = 3.4 +/- 0.3 mW/cm2. We chronically irradiated 10 Sk-1 albino hairless mice 5 times per week for 18 weeks, starting with 1.25 J/cm2, for 33 irradiation days, sequentially followed by 1.50 J/cm2 (34 days), 1.8 J/cm2 (10 days), 2.0 J/cm2 (22 days) to afford a total UVA dose of 154.3 J/cm2 over 99 irradiation days. Erythema was noted clinically by day 6, which persisted throughout the irradiation. During the irradiation period, some scaling, consistent with mild epidermal hyperplasia was noted during irradiation days 37-56. This response later regressed despite continued chronic irradiation. Hematoxylin and eosin examination immediately after the final irradiation revealed a mild inflammatory response, with some dermal restructuring. At the end of the experiment, no significant signs of epidermal hyperplasia or (pre)malignant lesions were seen, although some stratum corneum thickening was noted. Marked dermal collagen damage and moderate elastosis was also evident. We believe that the observed differences in results reported in previous studies are in large part due to differences in light sources and irradiation protocols.

Photochemistry of type I acid-soluble calf skin collagen: dependence on excitation wavelength.

Although previous studies have demonstrated that the predominant photochemistry of type I collagen under 254 nm irradiation may be attributed either to direct absorption by tyrosine/phenylalanine or to peptide bonds, direct collagen photochemistry via solar UV wavelengths is much more likely to involve several age- and tissue-related photolabile collagen fluorophores that absorb in the latter region. In this study, we compare and contrast results obtained from irradiation of a commercial preparation of acid-soluble calf skin type I collagen in solution with UVC (primarily 254 nm), UVA (335-400 nm) and broad-band solar-simulating radiation (SSR; 290-400 nm). Excitation spectroscopy and analysis of photochemically induced disappearance of fluorescence (fluorescence fading) indicates that this preparation has at least four photolabile fluorescent chromophores. In addition to tyrosine and L-3,4-dihydroxyphenylalanine, our sample contains two other fluorophores. Chromophore I, with emission maximum at 360 nm, appears to be derived from interacting aromatic moieties in close mutual proximity. Chromophore II, with broad emission at 430-435 nm, may be composed of one or more age-related molecules. Collagen fluorescence fading kinetics are sensitive to excitation wavelength and to conformation. Under UVC, chromophore I fluorescence disappears with second-order kinetics, indicating a reaction between two proximal like molecules. Adherence to second-order kinetics is abrogated by prior denaturation of the collagen sample. A new broad, weak fluorescence band at 400-420 nm, attributable to dityrosine, forms under UVC, but not under solar radiation. This band is photolabile to UVA and UVB wavelengths.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection against UV photocarcinogenesis by fabric materials.

BACKGROUND: Clothing fabrics have long been considered effective protection against short-term and long-term sun damage. Recently, special "highly UV-protective" fabrics have been developed specifically for photosensitive patients. OBJECTIVE: To determine if one such fabric will protect hairless mice against (pre)malignant lesions under conditions that will produce skin cancers through a typical summer fabric of moderate sun protection factor (SPF). METHODS: After prior determination of minimal erythemal dose, four sets of 10 animals were divided into the following groups: (A) dark control (no irradiation); (B) positive control (no fabric); (C) "typical summer" fabric (SPF = 6.5 +/- 1.0); and (D) "special" fabric (SPF > 30 across a broad spectrum). Mice were irradiated on the dorsal surface 5 days per week, with biweekly incremental increases (roughly 20% of the starting dose), for 12 weeks. Group B started at 6.3 J/cm2, with biweekly increases of 1.45 J/cm2 (total dose, 596 J/cm2); groups After irradiation, mice were observed for clinical and histologic signs of pre(malignant lesions for an additional 12 weeks. RESULTS: At the end of the 12-week irradiation period, nine mice in group B and six mice in group C had actinic keratosis-like lesions, whereas one mouse in group B and two mice in group C had squamous cell carcinoma. By week 24, all mice in groups B and C had squamous cell carcinoma. Mice in groups A and D showed no discernable reaction at any time. Biopsy specimens confirmed the clinical results. CONCLUSION: Typical summer clothing fabrics may offer inadequate protection against skin cancer and pose high risk to chronically photosensitive patients. The use of appropriate sun protective apparel should offer effective protection to photosensitive patients against short-term and long-term photodamage.

In vivo depigmentation by hydroxybenzene derivatives.

Certain mono- and dihydroxybenzene derivatives are selectively cytotoxic for melanocytes in vivo, and can cause depigmentation of skin and hair. We produced selective melanocytotoxicity/hair depigmentation in C57Bl mice by injection of 0.032-1.0% p-t-butylcatechol (tBC) or p-hydroxyanisole (MMEH) in physiological saline. No depigmentation occurred on injection of 3,4-dihydroxyphenylalanine (DOPA) or 3,4-dihydroxyphenylacetic acid (DOPAC). Light- and electron-microscopic examination of biopsy specimens taken from depigmented areas indicates selective melanocyte damage as early as 2 h post-injection. Melanocytes from anagen hair are most susceptible to depigmentation. All four compounds are substrates for tyrosinase, but only tBC and MMEH generate their respective isolable 1,2-benzoquinones, tBCQ and MMEHQ. These caused depigmentation in C57Bl mice to a comparable degree to the parent compounds. DOPA- and DOPAC-quinones (DOPAQ and DOPACQ) are not spectroscopically detectable in solution, suggesting extremely low steady-state levels of these compounds. The net observed rate of reaction of the respective 1,2-quinone with 300 microM bovine serum albumin (BSA) in vitro varies widely, with tBCQ >> MMEHQ = DOPACQ >> DOPAQ. The results are consistent with a mechanism involving attack of -SH on melanosomal proteins and/or enzymes by tyrosinase-generated 1,2-quinones. This mechanism evidently differs from that involved in in vitro hydroxybenzene melanocytotoxicity of melanoma cells, in which active oxygen intermediates generated by hydroxybenzene autoxidation play a significant role. The most reliable prognosticator of in vivo depigmentation appears to be the ability of the depigmenter to form a spectroscopically stable 1,2-quinone which is capable of reacting with protein -SH.

Dose-response of chronic ultraviolet exposure on epidermal forward scattering-absorption in SK-1 hairless mouse skin.

This work provides a dose-response model of UV-induced epidermal-stratum corneum thickening induced by irradiation at wavelength lambda. This model assumes that photobiochemical reaction(s) can give rise to hyperplasia in a manner which is predictable from a simple photochemical kinetic scheme. In this work, we derive an equation which predicts an approximately linear relationship between the logarithm of the increase in optical skin thickening measured at 320 nm (delta OD320) and total cumulative dose (DT) seen by the target cells in or near the basal layer. For each excitation wavelength lambda, the slope R(lambda) of the log delta OD320 vs DT plot is proportional to epsilon(lambda) phi rx, where epsilon(lambda) is the extinction coefficient for the target chromophore at excitation wavelength, and phi rx is the quantum yield for the photochemical reaction(s) leading to hyperplasia. Our data previously obtained from irradiation of SK-1 hairless mice with "monochromatic" UV wavebands at 280, 290, 300, 307 and 313 nm (Menter et al., 1988, Photochem. Photobiol. 47, 225-260.) and data from Sterenborg and van der Leun at 254 and 313 nm (1988, Photodermatology 5, 71-82) are in good agreement with this model, except for 254 and 280 nm excitation, which are greatly attenuated by epidermis-stratum corneum. For excitation at the latter wavelengths, "dark" regressive processes successfully compete with the "light" reaction(s) which lead to (pre)cancerous lesion. This difficulty notwithstanding, the "intrinsic" action spectrum for hyperplasia derived from these measurements indicates that the target chromophore preferentially absorbs in the UV-C region.(ABSTRACT TRUNCATED AT 250 WORDS)

Melanin accelerates the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH)

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultraviolet radiation-induced suppression of contact hypersensitivity in relation to padimate O and oxybenzone.

Contact hypersensitivity (CHS) in mice can be induced by cutaneous sensitization followed by elicitation via ear-painting with trinitrochlorobenzene (TNCB). This CHS reaction is systemic and can be suppressed by exposure of mice to suberythemogenic doses of 280-315 nm radiation. In this study, we investigated whether a commercially available water-resistant sunscreen, either SPF-6 or SPF-15, containing Padimate O (UVB absorber) and oxybenzone (UVA absorber), was effective in preventing systemic suppression of CHS induced by either FS36 sunlamp exposure or solar simulating radiation. We observed that these two sunscreen preparations were totally incapable of preventing the immunologic suppression of contact hypersensitivity by UV radiation. These results indicate that application of sunscreen does not retard the development of suppression of CHS following repeated UV exposure under conditions where erythema is not clinically observed. Thus, erythema may not be a good end point for assessing systemic immune suppression and its consequences.

Interaction of several mono- and dihydroxybenzene derivatives of various depigmenting potencies with L-3,4-dihydroxyphenylalanine-melanin.

Certain mono- and dihydroxybenzene derivatives cause depigmentation of skin and hair, and appear to be selectively cytotoxic for melanized pigment cells. As direct physical and/or chemical interaction between depigmenter (DP) and pigment melanin may play a role in depigmentation, we have carried out preliminary studies in model systems where such interactions may easily be separated from effects due to tyrosinase, melanosomal proteins, and other components. We have used synthetic L-3,4-hydroxyphenylalanine (L-DOPA)-melanin as a protein-free model pigment and potassium ferricyanide as a model electron acceptor. Compounds studied were catechol, 4-t-butylcatechol, 4-methylcatechol, 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid, hydroquinone, 4-methoxyphenol, 4-t-butylphenol, and 2,6, di-t-butyl-4-methylphenol (BHT) in 0.1 M phosphate buffer, pH 7.4. These compounds vary widely in their ability to depigment hair and skin. Ferricyanide reduction by DP in the presence and absence of melanin was monitored spectrophotometrically. The sparingly soluble BHT and 4-t-butylphenol did not reduce ferricyanide in the absence or presence of melanin. For the other compounds, kinetic analysis demonstrated direct interaction between each DP and melanin. Except for dihydroxyphenylacetic acid, reduction kinetics were consistent with a mechanism involving noninteractive binding of both DP and ferricyanide to melanin prior to coupled electron transfer through the melanin backbone. Kinetic analysis afforded KB, a thermodynamic constant (M-1) for DP-melanin binding, and k', a rate parameter (M s-1) for electron transfer. A dimensionless enhancement factor (EF) was defined as k'KB/ks, with ks a pseudo-first-order constant (s-1) for ferricyanide reduction in the absence of melanin. Depending on the reductant, melanin either retards (EF less than 1) or accelerates (EF greater than 1) the rate of ferricyanide reduction. There appears to be a direct relationship between EF and depigmenting potency. There is no relationship between depigmenting power and the ability per se of the DP to bind to melanin or to reduce ferricyanide.

Psoralens: a search for more effective derivatives for photochemotherapeutic regimens.

The objective of our studies under the National Toxicology Program on psoralens was to evaluate a new furocoumarin derivative that would be highly efficacious and yet possess little or no systemic toxicity while also having a short effective biologic half-life. In addition, this work allowed for the development of a test system for compound evaluation of various psoralen derivatives. A guinea pig model was used first, followed by definitive studies in hairless mice for evaluation of the phototoxic potentialities of 32 furocoumarins and 4 benzofuran derivatives. Rank order depends on whether the derivative is administered topically or orally; methyl furocoumarins were the most potent topical photosensitizers, but they were weak when orally administered. On the other hand, aminomethyl derivatives as a group were most potent of all the derivatives when orally administered but were mediocre topical photosensitizers. The standard for these studies, 8-methoxypsoralen (8-MOP), was mediocre in topical and oral regimens. Specific dose-response studies revealed the 5'-aminomethyl-4,4',8-trimethylpsoralen derivative to be six times more potent than 8-MOP. Moreover, the dose-response curve indicated that the response of the 5'-aminomethyl derivative is eliminated three to four times faster than is 8-MOP. No straightforward relationship between molecular structure and photosensitizing power was found. These results underscore the need for oral and topical evaluation of a given test sensitizer as well as for determination of the chemical nature, temporal distribution, and metabolic fate of its photochemically active form.

Effect of varying dose of UV radiation on mammalian skin: simulation of decreasing stratospheric ozone.

To better understand the dependence of the incidence of squamous cell carcinoma on changes in solar spectral distribution and dose regimen, we exposed SK-1 hairless mice to solar-simulating radiation (290-400 nm). Selective UV filtration was accomplished by passing this radiation through Schott WG-320 cutoff filters of 0, 0.5, 1.0, 2.0, and 3.0 mm thickness. Minimal erythema doses (MED) were determined for each filter combination. Starting with 0.5 and with 0.9 MED, groups of 20 mice were irradiated 5 days per week; this was increased by 20% increments (of the original dose) every 6th day for 40 days ("0.5 MED" and "0.9 MED" experimental groups, respectively). Other groups of mice were irradiated with the same incremental increases, starting at 6.5 J/cm2 ("equal dose" regimen). The salient results were: (1) shorter wavelength components appear to preferentially produce tumors; (2) resultant observable dose-response behavior for each regimen is a complicated function of concurrent "light" and "dark" reactions; (3) time-dose reciprocity is absent; and (4) there are no straightforward relationships among tumor efficiency, dose fractionation, and spectral distribution of excitation radiation. These results indicate that photocarcinogenesis is a dynamic process, in which events that result in tumor growth compete with those that cause tumor regression.

The rapid induction of cancers in the hairless mouse utilizing the principle of photoaugmentation.

We report a method for rapidly inducing cancer in the hairless mouse utilizing regimen in which an exposure to highly erythemogenic, but otherwise clinically noninjurious, dose of broad spectrum (290-400 nm) ultraviolet light is increased by 20% every 6th day. Clinical and histological observations reveal the presence of squamous cell cancer after as little as 18 days of irradiation. The rate of cancer induction is enhanced by the 320-400 nm component and this enhancement is shown to be a photoaugmentative effect. The results support the idea that stratum corneum and/or malpighian layer thickening produced in early stages of tumor induction tends to protect against the detrimental effects of UV radiation. Strict monitoring of both the spectral distribution and output of the radiation source is imperative for reproducible rates of tumor induction.

The interaction of L-DOPA melanin with p-tert-butylcatechol.

The interaction between the skin depigmenter 4-tert-butylcatechol (tBC) and L-DOPA melanin was qualitatively studied by means of spectroscopically monitoring the binding of tBC melanin as well as the oxidation of tBC in the presence and absence of melanin. Additionally, we assessed the quantitative effect of tBC on melanin's chemical reactivity by using the reduction of potassium ferricyanide as a redox marker. The kinetics of ferricyanide reduction in the ternary (melanin/tBC/ferricyanide) system are essentially different from those in each of the binary components (i.e., melanin/ferricyanide and tBC/ferricyanide). The experiments indicate that tBC can bind to melanin (KB = 3.8 X 10(3) M-1) and that melanin can act as a "catalyst-like" electron transfer agent which couples ferricyanide reduction with tBC oxidation (k' = 1.3 X 10(-6) mole min-1). These kinetic and thermodynamic parameters may provide a means of quantitatively comparing melanins obtained from different biological and pathological situations, and they may make possible an understanding of cutaneous depigmentation processes on the molecular level.