Free radical reduction in the human epidermis

0891-5849/89 $3.00+.00 © 1989 PergamonPressplc

Free Radical Biology & Medicine, Vol. 6, pp. 519-532, 1989

Printed in the USA. All fightsreserved.

Review Article FREE RADICAL REDUCTION IN THE HUMAN EPIDERMIS KARIN U. SCHALLREUTER*t and JOHN M. WOOD:~ *Department of Dermatology, University of Hamburg, Martinistrasse 52, D2000 Hamburg, FRG; :~Gray Freshwater Biological Institute, Department of Biochemistry, University of Minnesota, St. Paul, MN, U.S.A. (Received 8 March 1988; Revised and accepted 27 May 1988) A b s t r a c t - - T h e human epidermis presents the first line of defense against invading free radicals. Therefore, the surface of the skin must be equipped to deal with both the penetration of ultra-violet light as well as the neutralization of reactive photochemical products such as superoxide anion radical, hydrogen peroxide and especially hydroxyl radicals. Consequently, the human epidermis contains a variety of anti-oxidants to reduce oxygen radicals and hydrogen peroxide. The photochemical production of hydroxyl radicals, from both extracellular and intracellular hydrogen peroxide, is of special significance to the integrity of cells in the human epidermis. Recently, both biochemical and clinical studies on the healthy human population, and on patients with pigmentation disorders, suggested a connection between free radical defense by plasma membrane associated thioredoxin reductase and melanin biosynthesis. This research provided the first evidence for a direct relationship between free radical concentration and pigmentation. Furthermore, this system has been shown to be regulated by both extracellular and intraeellular calcium concentrations. Clinical studies show depigmentation disorders vitiligo and tyrosinase positive albinism (Hermansky-Pudlak syndrome) appear to have defective calcium uptake systems influencing both free radical defense and melanin biosynthesis.

Keywords--Free radical reduction, human epidermis, pigmentation disorders

INTRODUCTION

This research was supported in part by grants from the National Institute of Health AM 18101, the Mayo Foundation, the Marchionini-Stiftung, and the University of Hamburg. tAuthor to whom correspondence should be addressed. Dr. Karin Schallreuter was educated in Pharmacy, Greifswald, GDR. before studying medicine at the University of Hamburg, FRG. (1978-1984). She obtained her MD in 1984 and was awarded the Joseph Kimmig prize for her thesis on "The Mechanism of Quaternary Ammonium Compounds as Contact Allergens in Guinea Pigs". She held a studentship in Dermatology in the Mayo Clinic (1983) and completed her internship in Dermatology/Internal Medicine at the University of Minnesota and in Surgery at Altona Hospital, Hamburg in 1984. She was NIH-research associate at the University of Minnesota in Biochemistry/Dermatology (1985-1986). Currently she holds the position of Research Associate in Dermatology at the University of Hamburg FRG. She is author of 20 papers in the peer reviewed literature in the fields of Dermatology and Biochemistry. Professor Wood is a graduate in Biochemistry from Leeds University, England (1964), where he studied with the late Professor S. Dagley on the role of dioxygenases in aromatic metabolism. In 1966 he joined the faculty of the School of Chemical Sciences at the University of Illinois, Urbana. Between 1966-1974 he worked on the free radical mechanism of B,2-enzymes and on the mechanism of action of dioxygenases. He was the first to apply the technique of spin-labeling to B,2-enzymology in 1970-1972. In 1974 he moved to the University of Minnesota to direct the Gray Freshwater Biological Institute (1974-1979). He is currently Professor of Biochemistry at the University of Minnesota. Honors include: (1) the first recipient of the Synthetic Organic Chemistry Manufacturers Association gold medal for his work on methylmercury (1972); (2) Guggenheim Fellowship at Oxford (1972-1973); (3) American Chemical Society Zimmermann Award (1979); and (4) A member of the Editorial Board of Science (1982-1983).

The human e p i d e r m i s provides the o u t e r m o s t barrier against the invasion o f infectious agents, reactive electrophiles and free radicals. The surface o f the skin is e s p e c i a l l y vulnerable to d a m a g i n g free radicals generated by a n u m b e r o f p h y s i c a l and b i o l o g i c a l processes. P h o t o c h e m i c a l reactions with ultraviolet light, as well as ionizing radiation, p r o d u c e reactive o x y g e n intermediates such as s u p e r o x i d e anion radical (O2 ~), p e r o x i d e (02 =) and h y d r o x y l radicals ( . O H ) (ref. 1). Invasion o f the skin by p a t h o g e n i c bacteria, viruses and fungi attract neutrophils and m a c r o p h a g e s to inflamed areas and these cells p r o d u c e high local concentrations ofO27 through a respiratory burst involving a large increase in m o l e c u l a r o x y g e n consumption. 2 Physical burns or i s c h e m i a cause alterations in metabo l i s m c u l m i n a t i n g in the biosynthesis o f 02 ~ from the activity o f xanthine oxidase. 3 Even the p i g m e n t a t i o n o f human skin appears to p l a y a role in O2= and O2 ~ generation from molecular oxygen upon irradiation with ultraviolet light. F o r e x a m p l e , p h e o m e l a n i n extracted from human red hair has been shown to p r o d u c e more O2 ~ and 02 = upon irradiation with ultraviolet light than eumelanin from b l a c k hair. 4 H o w e v e r , it should be noted that the natural U V - a b s o r b i n g p o l y m e r s eume-

519

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K.U. SCHALLREUTERand J. M. WOOD

lanin and pheomelanin-contain intrinsic free radicals themselves which may trap soluble available radicals by coupling reactions? Also, the UV-filtration properties of the melanins clearly hold a preventative function to protect proliferating cells situated on the basal membrane of the epidermis and even in the dermis itself. For this reason a correlation exists between free radical defense in the epidermis and skin pigmentation, with the more pigmented individuals yielding a more effective protection against UV-mediated skin damage. The biosynthesis of the melanins occurs in melanosomes which are situated in the basal and supra-basal cells of the epidermis. Melanogenesis in these organelles depends on the regulation of activity of the copper-containing enzyme tyrosinase. 6 Thus the activity of tyrosinase in human skin varies with the individual skin type. 7 An assessment of those free radical scavenging mechanisms known to be available in the outermost living (stratum spinosum and stratum granuiosum) and dead (stratum corneum) layers of the human epidermis are the subjects of this review. Special emphasis has been given to reactions occurring in the plasma membranes of keratinocytes and melanocytes. These free radical reduction reactions take place before the intervention of the melanins) FREE RADICAL BIOCHEMISTRY IN THE HUMAN EPIDERMIS

Generation of superoxide anion radical The production of O2: in the epidermis may occur by a large number of chemical and biochemical mechanisms 9,~° (Scheme l). These include: (a) ionizing radiation and ultraviolet radiation under aerobic conditions; (b) hyperbaric oxygen; (c) photosensitizing agents (e.g., psoralens and porphyrins); (d) enzymes and coordination complexes containing transition metals in their lower oxidation states; (e) infiltration of polymorphonuclear leukocytes in inflammatory skin

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Scheme 1. The synthesis of 02- extracellularly: (a) by UV-photochemical reactions and (b) by the phagocyte oxidase system on the plasma membranes of macrophages, and intracellularly: (a) UV-light and pheomelanin, (b) from hypoxanthine/xanthine oxidase, and (c) from gratuitous reactions with reduced transition metal complexes.

diseases; (f) burn-induced deamination of adenine to produce hypoxanthine which acts as a substrate for xanthine oxidase leading to the production of uric acid; (g) photochemical production from pheomelanins. The mechanisms described above can lead to the generation of 02; both extracellularly and intracellularly. Some of the reactions are very specific whereas others are gratuitous. The reaction of molecular oxygen with photochemical mediators or with transition-metal complexes are regarded as circumstantial and depend on oxygen concentrations, UV intensity, and cellular redox conditions in the epidermis. Biochemical mechanisms such as the xanthine and phagocyte NADPH oxidase reactions depend on the availability of hypoxanthine and on epidermal infiltration by polymorphonuclear leukocytes respectively. In all cases molecular oxygen functions as a single electron acceptor to produce 02 ~. Recently the mechanism of 02 ~ biosynthesis by phagocyte NADPH oxidase, and its regulation by divalent cations (Mg ÷* or Ca ÷÷) on the outside of the plasma membranes of macrophages has been elucidatedfl Electrons flow from reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the cytosol to an inner-membrane associated flavoprotein, and then to an outer-membrane associated cytochrome b558, and finally to molecular oxygen. This reaction causes a burst of oxygen uptake leading to a rapid increase in extracellular 02 ~- concentration. Enzyme activity has been shown to be regulated by the extracellular concentrations of Mg ÷÷ or Ca ++. Thus, these fast exchange ions play an important regulatory function in 02 = metabolism. The importance of Ca ÷+ in free radical defense and pigmentation has been described later in this review.

Reactions with superoxide anion radical As with its formation, the metabolism o f O 2- o c c u r s through a variety of chemical and biochemical processes. Since 02 = has been shown to be synthesized both extracellularly and intracellularly; mechanisms for its metabolism exist both on plasma membranes and in the interior of cells in the epidermis. Scheme I1 presents five alternative mechanisms for O2 ~ metabolism: (a) Membrane-associated thioredoxin reductase (TR) reduces 2 O2- to O2 = and water outside the cell; this enzyme is subject to allosteric inhibition by Ca ++ (refs. 12-20); (b) Superoxide dismutases (SOD) produce 02- and oxygen from the dismutation of 2 O2 = in the cytosol; 2~ (c) Indoleamine 2.3 Dioxygenase (I2, 3D) uses O2 ~ as a specific substrate for the oxidation of L tryptophan to N-formyl kynurenine; 22 (d) metalcomplexes, including metallo enzymes, containing

Free radicals in skin

202

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Scheme II. The metabolism of O2~ extracellularly on keratinocytes by thioredoxin reductase (TR), and intracellularly by (a) superoxide dismutases (SOD's); (b) indoleamine 2,3 dioxygenase (12,3D) which uses O2: as a substrate for the oxidation of L-tryptophan to Nformylkynurenine; (c) a reaction with intrinsic free radicals on melanin M', to give organoperoxide products (M--O---O-); and (d) by gratuitous oxidation reactions with oxidized transition metal complexes.

transition metals in their higher oxidation states are capable of the gratuitous oxidation of 02 ~ to molecular oxygen.~.~J This reaction depends on the redox-status of the cell and is reversible. (e.g., Fe Ill -I- 02 ~ Fe n + 0 2 ) and (e) Intrinsic free radicals in melanin may trap 02- to produce reactive organo-peroxy intermediates on this polymer. 5 A considerable amount of knowledge has accumulated on the importance of TR, SOD, and I2, 3D as free radical scavengers. The discovery of membraneassociated TR as a free radical trap at the surface of the skin depended on the synthesis of a suitable model radical substrate, exhibiting the important properties of surfactant and plasma-membrane impermeability. 17"~8 A spin-labeled analog of benzalkonium bromide has been synthesized which does not penetrate the plasma membranes of keratinocytes, melanocytes and melanoma cells.~S This nitroxide radical substrate has been shown to be selectively reduced at the outer cell surface to a secondary amine.~2 Furthermore O j has been found to be a competitive substrate for nitroxide radical reduction. ~2Since nitroxide radicals are known to be easily reduced to the hydroxylamine product by single electron transfer processes both inside plasma-membranes 23'24 and inside the cell by small molecules and enzymes of the cytosol and mitochondria; 24; it has been important to determine whether the reduction of the spin-labeled quaternary ammonium compound to a secondary amine occurred on a specific active site. Initially it was discovered that this spinlabel reduction was catalyzed by a thioprotein (i.e., thiolate active site), because activity was totally in-

521

hibited by parachloromercuribenzoate (PCMB) and Nethylmaleimide (NEM). 13'16 This inhibition was demonstrated at the surface of skin, on keratinocytes and melanocytes, and on purified T R ' s themselves. These findings established a quantitative role for a plasmamembrane associated thioprotein in the reduction of the spin labeled quaternary ammonium substrate. The identification of TR as this protein has been obtained from the following direct and indirect evidence. (1) TR has been purified from plasma membranes of human keratinocytes and from human metastatic melanotic melanoma cell membranes.~3.2° (2) Rozell et al. 25 and Hansson et al. 26 prepared fluorescent antibodies to TR and its natural electron acceptor thioredoxin (T) isolated from rat liver, and showed that these proteins can be plasma membrane associated. (3) The reduction of spin labeled quaternary ammonium substrate has been competitively inhibited by the natural substrate thioredoxin. ~2 (4) TR has been shown to be inhibited at the surface of the skin, on keratinocytes, melanocytes and melanoma cells, and on purified T R ' s by: (a) thioprotein inhibitors PCMB and NEM; 13'16 (b) active-site labeling with spin-labeled maleimide; ~6(c) allosteric inhibition by C a + + ; 16 (d) activities on cells were inhibited with fetal calf serum both in the culture medium and in bioassays; ~3 (e) by NADP ÷, T R ' s own oxidized coenzyme; j3 (f) by anthralin; 27 and (g) by azelaic acid and other saturated dicarboxylic acids (C9C~2). j9 Therefore, the specificity of the TR bioassay on human skin and on the surface of epidermal cells has been satisfied by eight properties common to purified TR. Despite this direct, and indirect proof, it was recognized that glutathione reductase (GR) has very similar properties to TR. GR has a similar activity to TR. Both enzymes are flavoproteins and in both cases electrons flow from NADPH to a protein bound FAD molecule which reduces a second disulfide active site. 28,29The dithiolate active site in yeast GR has been shown to be separated by four amino acid residues (valasp-val-gly) whereas E-coli TR has only two residues (ala-thr) between the cysteine thiolates. 29 O'Donnell and Williams have shown that TR has been a much stronger nucleophile than GR, with a pKA for the thiol to the thiolate equilibrium of 6.98; two units lower than the same acid base equilibrium in GR. 28The stronger nucleophilicity of TR allows complete reduction of the spin-labeled quaternary ammonium-substrate to a secondary amine. GR will not catalyze this reduction reaction.~2 Taken together, all of these results indicate that the spin-labeled quaternary ammonium substrate may be safely used for a direct assay of TR in clinical studies even in the complex biological matrix of human skin. The reduction of O2 ~ to 02 = has been shown to be a reaction common to most flavoproteins, and the

522

K.U. SCHALLREUTERand J. M. WOOD

reduction of O2= to water by the dithiolate active site of TR may be viewed as a similar mechanism to that proposed for the selenohydryl group of glutathione peroxidase (GP). With both TR and GP the mechanism for S - - S and S - - S e bond formation in their active sites must be kinetically favored over oxidation of the respective sulfhydryl and selenohydryl groups by O2 = (Scheme III). In 1985 Chen and McLaughlin 23 reported the reduction of 5-doxyl stearic acid, a plasma membrane probe, in mouse neuroblastoma cells. In these experiments it was assumed that the GR-glutathione system was responsible for this NEM-inhibited reaction. The system was never isolated and characterized, nor was the product analyzed to determine whether reduction to the hydroxylamine or secondary amine occurred. The TR-thioredoxin system was never considered in this study even though these thioproteins are known to be plasma membrane associated. In reactions concerned with cellular redox-balance, the TR-thioredoxin system has been mostly neglected and the GR-glutathione antioxidant mechanism invoked.

Skin photosensitizing agents such as the psoralens produce O j which can be scavenged by the addition of superoxide dismutase " i n v i v o " at the surface of human skin. 3° Therefore, it has been important to examine the contribution made by SOD's to free radical defense in the epidermis. Human skin contains both the CuZn-SOD and the Mn-SOD. 21 SOD activities in the human epidermis has been shown to be considerably lower than that of heart, kidney, or liver. 21 This result has been surprising when one considers the potential for exposure to high concentrations of 02 ~ from photochemical reactions in the epidermis. These results suggest that enzymes much as TR (extracellular) and the GR-GP system (intracellular) may be more important free radical scavengers in the epidermis than the SOD's. It should be emphasized that SOD has the disadvantage of producing intracellular 02 = as a product and this may be readily converted to .OH by UVphotochemistry,l whereas light protected organs, such as liver, do not face this kinetically favorable light reaction pathway. I2,3D differs from other dioxygenases 31 in its uti-

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Scheme III. Proposed reaction pathway for the reduction of 02 = to peroxide and water by TR.

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Free radicals in skin lization of 027 rather than dioxygen as a preferred substrate. This dioxygenase has been characterized as a heme-containing enzyme with a molecular weight of 42,000 daltons. 22 Since O2 ~ has been shown to be a substrate for I2,3D, its enzyme activity has been inhibited by SOD but stimulated by 027 production from infiltrating polymorphonuclear leukocytes. 22 Enzyme activity has been induced by viral infections, bacterial endotoxin and by ~/interferon. 22 ~/interferon induced I2,3D 20 to 30 fold in 48 hours after injection into mice. 22 A 120 fold increase in the level of I2,3D was measured in the lungs of mice after the eleventh day of viral infection; with levels returning to normal after the virus disappeared. 22Osamu Hayaishi has developed a most interesting hypothesis in an attempt to explain the above findings. Tissues invaded by viruses or bacteria invoke an inflammatory response causing the infiltration of lymphocytes and leucocytes. A local respiratory burst would cause an increase in the concentration of O2 ~ with increased ",/interferon levels at inflammatory loci. These conditions have been shown to increase levels of I2,3D which depletes the L tryptophan pool. In fact the metabolism of L-tryptophan does occur under these conditions leading to increased kynurenine in the blood and urine of patients. 22 Since L tryptophan functions as an essential amino acid for viral replication and bacterial growth; its removal would be expected to prevent the spread of local infections. Another appealing aspect of this hypothesis has been a biochemical rationale for the mode of action of ~/ interferon. 22

The generation of hydrogen peroxide The chemical reactivity, and therefore the toxicity, of 02 ~ resides in its rapid conversion to more cytotoxic chemical species such as 02 = and -OH. The reduction of 027 to 02 = is catalyzed by both specific and gratuitous systems. Both SOD and TR produce 02 = from O2~-; also intrinsic free radicals on melanin as well as other single electron donors, such as reduced transition metal complexes, catalyze the gratuitous formation of O2 = (e.g., 027 "1- Fe" ~ Fe In + 02=). Cells in the epidermis have evolved extracellular and intracellular mechanisms for the metabolism of O2=. This has been the case for both hydrogen peroxide and other organoperoxides. 32

Reactions with hydrogen peroxide Reactions with 02 = occur outside cells of the epidermis, in the stratum corneum, and inside keratinocytes. In the stratum corneum thiol groups from pro-

523

tein-bound cysteinyl residues in keratins have been shown to be important traps for O2 = (ref. 32). A cysteine-rich protein has been purified by Tezuka and Takahashi which produces malondialdehyde by lipid peroxide reduction. This protein has been found to have a molecular weight of 48,000 daltons and contained 4.3% cystine residues or 40 cysteine residues/molecule. 32 The oxidation of these cysteinyl residues to disulfides represents an effective 02 = trap. In addition to this system, the extracellular matrix of the stratum corneum has been shown to contain cysteinyl peroxidase which utilized 02 = in the oxidation of disulfides to cysteic acid residues 33 (Scheme IV). Inside keratinocytes two biochemical pathways have been discovered for the metabolism ofO2=: (a) catalase and (b) the G R / G P system. The selenohydryl group at the active site of GP emphasized the importance of the bioavailability of the ultra-trace element selenium in human nutrition, and probably explained the rationale for evolutionary duplication of enzyme systems designed for the removal of 02 =. Extracellular and Intracellular metabolism of 02 = has been essential to the prevention of .OH synthesis by nonspecific Fenton Chemistry or by UV-mediated photochemistry. In fact all of the oxygen biochemistry described so far can be associated with detoxication mechanisms designed to prevent .OH synthesis. These radicals have been found to cause membrane lysis, DNA-alterations, protein damage, and thiol oxidation through covalent binding.

r--l SH SH

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H2O2"-~ LIPIDPEROXIOATIO~N H20~'~~O2+

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Scheme IV. The metabolismof hydrogen peroxide in the epidermis extracellularly by: (a) TR reduces O2= to water. (b) High cysteinyl protein HCP reduces peroxide to water by oxidizing dithiol to disulfide bonds in this protein which contains 40 cysteine residues. (c) Cysteinyl Peroxidase (CP) oxidizes cysteine thiols to cysteic acid residues. (d) UV-light photolyzes 02= to give damaging -OH radicals. Intracellulary02 = is metabolized by (a) catalase; (b) the glutathione peroxidase (GP)/glutathione reductase (GR) system; and (c) by gratuitous reactions with reduced transition metal complex to give damaging -OH radicals.

524

K . U . SCHALLREUTERand J. M. WOOD FREE RADICAL REACTIONS WITH PLASMA M E M B R A N E S OF H U M A N K E R A T I N O C Y T E S , M E L A N O C Y T E S AND M E L A N O M A C E L L S

TR activities have been determined by using spinlabeled quaternary ammonium substrate at the surface of cells harvested from human keratinocyte, melanocyte, and melanoma cell cultures. Specific activities have been determined as the decrease in nitroxide radical signal/10 minutes/unit of cell protein. '3 In order to perform meaningful comparative studies of enzyme activity, cells have to be established in medium without fetal calf serum, since calf serum inactivates TR. n3 Rapidly proliferating cells in culture media containing growth factors always yielded low TR activities, and cells grown with 2.0 × 10 -3 M Ca ++ have been approximately half as active as cells grown with 0.1 × 10 -3 M C a + + . j3 Ca ++ has been shown to be an allosteric inhibitor of TR both " i n vitro" and " i n v i v o . " ~6 Maximum TR activities have been measured for cells allowed to differentiate and stratify in medium containing 0.1 x 10 -3 M Ca ++. Melanoma cells yielded double the activity in TR over control cultures of melanocytes, and melanocytes revealed double the activity of keratinocytes. ~9These findings have been consistent with the fluorescent antibody studies of Rozell et al. 25"26who showed moderate to high immunofluorescent labeling of plasma-membrane associated TR on rat melanocytes. Muglia et al. 35 demonstrated that melanocytes had a special sensitivity to 02- toxicity compared to keratinocytes yielding no detectable levels of catalase activity. The increased level of TR on melanocytes may compensate for the absence of catalase. Keratinocytes established from patients with hypopigmentation disorders vitiligo and from tyrosinase positive albinos with Hermansky-Pudlak syndrome (HPS) showed low TR activities. 36'37

F R E E R A D I C A L DEFENSE AND P I G M E N T A T I O N IN T H E H U M A N E P I D E R M I S

A biochemical model for free radical defense and the regulation of melanogenesis An enzyme model system has been developed for the regulation of tyrosinase by the TR/T electron transfer system by using purified TR and T from Escherichia coli and mushroom tyrosinase respectively (Scheme V). ~2The model is based on the concept of competition for electrons from NADPH/TR by the two substrates, O2: extracellularly, and oxidized T intracellularly. '2 This switch for electron flow predicts that UV-generated free radicals should be preferentially reduced by membrane associated TR on the plasma membrane

A MODEL FOR THE DEFENSEAGAINST FREE RADICAL ATTACK BY THE THIOREDOXIN REDUCTASE- THIOREDOXIN SYSTEM LIGHT STIMULATED

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leaving intracellularly T in an oxidized state and thus preventing the inhibition of tyrosinase. In fact oxidized T had no inhibitory effect on tyrosinase.'2 However, in the absence of UV-generated radicals, TR preferentially reduces T, and this reduced thioprotein has been shown to inhibit tyrosinase by forming a stable biscysteinate complex with one of the active site copper atoms.'6 The active site of met-tyrosinase is composed of two tetragonal Cu[I complexes, bridged by a ligand from the protein, which allows spin-pairing between the two copper centers to give a diamagnetic complex with a copper-copper distance of 3.4 Angstrom. 38'39 Oxytyrosinase has a/z peroxo-bridge between the copper atoms forcing them further apart to 3.63 Angstrom. 38 The tyrosinase copper active site is much more accessible to large molecules than the similar active site in hemocyanin. 38 Reduced thioredoxin appears to inhibit tyrosinase by a similar mechanism to Lmimosine 4° by forming a complex with one of the Cu x~ centers yielding a paramagnetic product showing large rhombic distortion and a four line splitting pattern in the perpendicular region as determined by low temperature ESR. 38 However, the nature of the bridging ligand, and the identification of one of the bases in the

Free radicals in skin CUA site of tyrosinase remain unresolved and therefore an exact structure for the reduced thioredoxin-tyrosinase complex cannot be presented at this time. We assume an interaction between reduced thioredoxin and the CuA center because the CuB center is fully coordinated with 3 histidine ligands from the protein. This protein-protein interaction between reduced thioredoxin may be a crucial factor in the regulation of melanin biosynthesis. Consequently we proposed that the activity of membrane-associated TR at the surface of the skin should vary in different pigmented individuals from the normal healthy human population. A group of 40 clinically healthy human volunteers with different skin types (I-VI) Fitzpatrick Classificatio# 4 were examined for TR activity. The results of this study showed a clear correlation between TR activity and skin pigmentation with type I skin yielding 6.1 - 3.5 units compared to 29.7 --- 4.8 for type VI. These results provided the first clinical evidence in support of the enzyme model, j2A4 However, a more stringent test for a connection between TR, free radical defense, and pigmentation resided in the examination of patients with depigmentation disorders such as vitiligo and albinism.

Clinical studies on patients with hypopigmentation disorders vitiligo and tyrosinase positive albinos with Hermansky-Pudlak syndrome Tyrosinase positive albinos with HPS. The initial purpose of this clinical study has been to test the enzyme model for the regulation of tyrosinase. HPS homozygotes do have tyrosinase but this enzyme activity is rarely expressed. Based on the enzyme model it was anticipated that tyrosinase positive albino's with HPS should have low TR activities in the reduction of the free radical spin-labeled substrate. To examine TR activity in this hypopigmentation disorder, the HPS population in Puerto Rico was chosen for 4 reasons: (a) These albinos are tyrosinase positive, but their melanin biosynthesis seems to be defective. (b) Normal family members present skin type IV to VI, and therefore large differences in TR activities were anticipated between albinos and pigmented individuals. (c) The prevalence of this genetic disorder is 1 in 2,000, and (d) many large families were available for studies of this disease. HPS has been classified as an autosomal recessive disorder with a tyrosinase positive form of oculocutaneous albinism. Patients have a mild bleeding diathesis due to storage pool deficient platelets, and older patients accumulate a lypofuchsin-like (ceroid) material in different tissues. 42 The absence of platelet dense bodies, the storage

525

organelles for calcium, non-metabolic ATP and ADP, and serotonin; has been the major basis for disease diagnosis in HPS.42 Recently Schallreuter and Witkop 4~ have shown that TR activity may also provide a very precise method for diagnosis of HPS and also for the identification of obligate and putative heterozygotes in the Puerto Rican population. Forty five individuals from seven families were tested including 12 homozygotes, 9 obligate heterozygotes and 24 unclassified probands. Additionally seven separate non-kindred HPS-homozygotes were examined. Figure la shows that all but one HPS homozygote exhibited low TR activities, and this exception was an unusual dark-haired/dark skinned HPS patient with platelet pool deficiency and a nystagmus characteristic of this type of oculocutaneous albinism. All of the fair-skinned, fair-haired homozygotes showed low rates for the reduction of the spinlabeled substrate. TR activity was somewhat lower than expected in 8 obligate heterozygotes and in 12 unclassified family members. Ten subjects showed normal TR activities according to each individuals skin type. Differences between TR values, based on individual skin types, and the experimentally observed values for heterozygotes and normal family members are presented in Figure lb. The separation of the HPS heterozygotes TR activity (x = 17.1 units) from the normal family members TR activity (x = 25.5 units) has been sufficient to detect carriers of the HPS genetic defect in the normally pigmented population. A most puzzling aspect of this clinical study resided in the discovery that normally pigmented HPS heterozygotes presented lower TR activities than expected, even though their TR was generally higher than in their HPShomozygote kindred. The answer to this question emerged when the regulation of the TR/T/tyrosinase cascade has been found to be due to allosteric inhibition by calcium. Cellular and molecular evidence is now available to show that HPS may be caused by either a defective Ca + + uptake system or by an inability of HPS cells to retain steady state levels of this fast exchange ion. 37 Despite the complications caused by Ca ++ regulation of TR, these clinical data yield further support for the molecular model connecting TR and melanin biosynthesis. 12

Vitiligo. A clinical study of patients with vitiligo seemed appropriate to test the molecular model, because patients with this disease present both pigmented and regions of totally depigmented skin. The etiology of this depigmentation disorder is currently not understood. There has been circumstantial evidence that environmental factors, such as an incident of excessive solar exposure, or an emotionally traumatic episode, triggered the onset of vitiligo, s It has been shown that

526

K . U . SCHALLREUTERand J. M. Wood

DIFFERENCE IN T R UNITS BY SKIN TYPE Normal Value minus Patient Value

UNITS OF T R ACTIVITY DIRECT ASSAY

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-6 Fig. la. A comparison of TR activities in the Puerto Rican HPS population.

Fig. lb. Identification of putative heterozygotes with HPS in seven Puerto Rican families.

Free radicals in skin the regional loss of melanocytes appears to be caused by an auto-immune response, although it has not been determined whether this immunologic reaction was the cause or the effect of the disease. 43 Certainly vitiligo may be induced chemically due to excessive occupational exposure to electrophiles such as phenols and quinones, suggesting an environmental/biochemical etiology for the disease. 44 Ten patients with vitiligo were tested for TR activity on their pigmented and depigmented skin. 14Four conclusions were drawn from this analysis: (a) In each individual case depigmented skin was 30-80% lower in TR activity compared to adjacent pigmented skin biopsies. (b) Pigmented skin was consistently more active in TR than those activities expected for each individual's skin type. (c) TR activities in depigmented skin were closer to the expected skin type values than pigmented skin. (d) Two cases yielded major differences in activity between pigmented and depigmented skin, that is ( - 8 0 % ) , whereas eight cases showed only ( = 3 0 % ) difference; suggesting the possibility of subclasses, or stages, for progression of the disease. Subsequent work with cell cultures of keratinocytes indicated that, as with HPS, vitiligo has a defect in calcium transport. 36 For each individual case studied the results on vitiligo support our molecular model. However, an unexpected result in the clinical study of vitiligo has been the observation that pigmented skin appears to be hyperactive in TR over each individuals expected skin type value. One consequence of increased TR-activity at the surface of the skin would be an increase in the concentration of it's reaction product H202. This increase in H202 would be expected to cause an increase in .OH either through UV-photochemistry or by reactions with metal-complexes, l Membrane damage and cell lysis in the epidermis has been verified in the pigmented skin of patients with vitiligo. 45'46These histological changes may be caused by .OH radical damage. Also, since melanocytes appear to lack catalase activity 35 and show a special sensitivity to H202, it is possible that increased levels of H202 in the epidermis may oxidize proteins at the surface of melanocytes sufficient for the induction of the observed auto-immune response. 43

The regulation of free radical defense and melanin biosynthesis by Ca +÷ Preliminary experiments with serum-free cultures of keratinocytes and melanocytes showed that TR-activity depends on the extracellular Ca + + concentration. 13The first evidence for a direct effect of Ca ++ on enzyme activity came from a series of experiments where keratinocyte were grown in medium containing 0.1 ×

527

10 -3 M Ca ++ harvested, and switched to 2.0 × 10 -3 M Ca ++ medium 20 minutes prior to assay. The TR activity dropped from 28.6 units in 0.1 × 10 -3 M Ca ++, to 2.7 units in 2.0 × 10 -3 M of this fast exchange ion. 15 TR activities were measured in the presence and absense of Ca ++ at the surface of the skin, on melanoma biopsies, at the surface of keratinocytes and melanocytes and on purified T R ' s from Escherichia coli and human keratinocytes. 15 In all cases Ca ++ was shown to inhibit the reduction of the spin labelled substrate in a concentration dependent manner.15 The allosteric nature of this regulation of TR activity by Ca ++ was proved by using an active site directed inhibitor 4-maleimido-Ca + + tempo (Figure 2). O'Donnell and Wiltiams 47 had previously shown that both thiolate active sites can be labelled with 14C Nethylmaleimide, this was confirmed by showing that both active sites can be labelled with 4 maleimidotempo to give a fully inhibited TR (ref. 15) (Figure 2). The two labeled thiolate groups on TR can be distinguished by ESR, one site yielding a more immobilized spin label than the other. J5 Upon the addition of Ca ++ to this enzyme inhibitor complex the more immobilized nitroxide radical has been selectively reduced by the enzyme-bound FADH2 to give an ESR-silent product (presumably the hydroxylamine product). The less immobilized spin label was not reduced either in the presence or absence of Ca ++ (ref. 15). It appears that the addition of Ca ++ caused a sufficient conformational change at the enzyme active site bringing the more immobilized label closer to the FADH2 moeity to allow a Ca + +-dependent electron transfer reaction. The regulation of TR activity by extracellular Ca + + concentrations suggested that the uptake of Ca ++ by ATPdependent plasma membrane channels may be important to free radical defense and also to the regulation of pigmentation through feedback on tyrosinase. To test this hypothesis, keratinocytes were established from HPS homozygotes, HPS obligate heterozygotes and from normal family members. Keratinocytes from four HPS homozygotes were grown in synthetic culture media containing different Ca + ÷ concentrations, and TR was shown to be especially sensitive to inhibition by Ca + ÷ compared to controls (Table 1). Isotopic 45Ca+ + has been used to follow the fate of this regulatory ion in keratinocytes established from one HPS homozygote, one HPS obligate heterozygote and one normal family member. The following conclusions could be drawn from these experiments: (1) Keratinocytes from the normal Puerto Rican family member transported 45Ca++ rapidly to a steady state with low extracellular Ca +÷ and high intracellular Ca ++ (2) HPS homozygous cells transported Ca +÷ slowly and also appeared to leak Ca ++ yielding high

528

K . U . SCHALLREUTERand J. M. WOOD

Fig. 2. (a) ESR-spectrum of free 4-maleimido-tempo. (b) ESR-spectrum of 4-maleimido-tempo covalently attached to the two thiolate groups in the active site of TR. One of the labeled thiolate inhibitor complexes is more immobilized than the other.

extracellular and low intracellular concentrations. (3) HPS obligate heterozygous cells showed an intermediate uptake rate for 45Ca+ ÷ between (1) and (2) above, yielding approximately equal concentrations of Ca +÷ inside and outside cells. The results from these " i n vitro" experiments support the molecular model for the regulation of tyrosinase by TR if one considers the regulation of this enzyme by extracellular Ca ÷ ÷ concentration. Increased extracellular Ca ++ in HPS homozygotes may be caused by either (a) a defective Ca + ÷ -ATP-ase uptake system or (b) an inability of HPS cells to retain Ca + + (i. e., cells leaky to Ca + + ).37 The platelet dense body depletion found in HPS may also reflect an inability of these cells to retain Ca +÷ . Also, since the TR system together with melanin biosynthesis are crucial to free radical defense in the epidermis, these results provide a rationale for increased solar susceptibility in HPS patients compared to normal healthy family members. 37

Table 1. The Regulation of TR Activity by Ca ++ in Keratinocytes of HPS Homozygotes (n = 4) and Controls

Keratinocytes

Homozygotes

Controls Adult Neonatal

TR Activity in Medium with 0.1 mM Ca ÷+a

Activity in Medium with 2.0 mM Ca ++

Keratinocyte cell cultures from pigmented and depigmented skin from a patient with vitiligo were established in serum-free culture medium containing different Ca ++ concentrations. ~3 Both TR activities for these keratinocytes revealed no significant differences in 0.1 × 10 -3 M Ca ++, compared to normal control cells, whereas keratinocytes grown in 2.0 × 10 -3 M Ca +÷ yielded extremely low TR activities. (Table 2). These results differ significantly from those observed for depigmented skin in HPS; (1) Much higher concentrations of Ca + ÷ are required to inhibit TR on vitiliginous keratinocytes compared to HPS homozygous keratinocytes (2) 45Ca ++ uptake showed that vitiliginous keratinocytes reach the same intracellular steady state for Ca ++ as healthy controls, or pigmented skin controls, whereas HPS homozygous keratinocytes leak Ca ++ (ref. 36). While the results on HPS suggest a breakdown in free radical defense caused by Ca ++ regulation of TR, this conclusion does not apply to vitiligo, because TR activities on depigmented skin are closer to normal skin

Table 2. Specific Activities for Thioredoxin Reductase at the Surface of Differentiated Adult Human Keratinocytes from a Vitiligo Donor (J.M.)

Age

Sex

1 2 3 4

26 39 44 27

M M F F

9.5 11.0 12.0 6.0

3.0 4.0 6.0 3.0

Skin Site

Medium Ca ++

42 --

F M

8.50 40.0

40.0 17.0

Normal Vitiliginous Normal Vitiliginous

0.1 mM

1 2

aTR activity (units) decrease in amplitude of nitroxide radical signal/OD280! 10 minutes.

2

mM

Rates/10 Min in Differentiated Keratinocytes/GF ( - )/a 66 70 8 6

aGF = Growth factors absent ( - ) in the culture medium.

Free radicals in skin type values in vitiligo patients, and extracellular Ca + ÷ bioaccumulation has been insufficient to explain depigmentation caused by inhibition of the TR/T/tyrosinase cascade. 12 In conclusion, HPS appears to be caused by a defect in the Ca÷÷-transport system. Vitiliginous keratinocytes do have a decrease in Ca÷÷-uptake, but despite this defect, intracellular Ca ÷ ÷ reached the same steady state in cells established from pigmented and depigmented skin. These results indicate that other factors must be important in the depigmentation process and the loss of melanocytes in vitiligo.

The mechanism of action of the depigmenting drug azelaic acid Azelaic acid has been reported to be a competitive inhibitor of tyrosinase and therefore has been assumed to function as a depigmenting agent by preventing melanin biosynthesis directly, as Due to its depigmenting properties and its low toxicity azelaic acid has been used in the treatment of hyperpigmentation disorders lentigo maligna and melasma. 49 Recently this drug has caused some controversy in the literature regarding the treatment of lentigo maligna because some patients treated with azelaic acid progressed to lentigo maligna melanoma. 5°,51 Azelaic acid and other saturated dicarboxylic acids (C6-CI2) were shown to be weak competitive inhibitors of tyrosinase when L tyrosine was used as a substrate. 52 Experiments with catechol as substrate showed no inhibition of tyrosinase activity, and the monomethyl ester of azelaic acid was found to be only half as inhibitory as azelaic acid itself (i.e., K~ azelaic acid = 2.73 × 10 -3 M and Kt azelaic acid monomethyl ester = 5.24 x 10 -3 M ) . 53 These results suggested that azelaic acid functions as a competitive inhibitor by reacting with the a carboxylate binding site for L-tyrosine on tyrosinase. 53 The kinetic data indicated that at least 10 -3 M azelaic acid must be transported through cells and into melanosomes for effective direct inhibition of tyrosinase. Furthermore, experiments with 3H dodecandioic acid (CI2), a more effective inhibitor of tyrosinase, failed to show the incorporation of this dicarboxylic acid in melanos o m e s . 5a Intracellular concentrations as high a s 10 -3 M azelaic acid inhibited DNA synthesis, and was shown to be cytotoxic by disrupting mitochondria. 55 Taken together, these results suggested that it was most unlikely that azelaic acid could reach a high enough intracellular concentration to inhibit tyrosinase directly and prevent melanin biosynthesis without being cytotoxic. Recently it has been shown that azelaic acid inhibits membrane associated TR (Kt = 12.5 × 10 -6 M).I9 Based on the 200-fold difference in K~ for this

529

inhibition reaction, an indirect effect on TR, leading to a feedback on tyrosinase appears to be a more plausible mechanism for the depigmenting property of azelaic acid. The KI for azelaic acid on membrane-associated TR has been well below the cytotoxic concentration measured for this saturated dicarboxylic acid.55 Scheme VI presents the alternate mechanisms for inhibition of tyrosinase by azelaic acid indicating the importance of kinetic parameters. lac azelaic acid has been used to study the mechanism of TR inhibition by this saturated diacid. The stoichiometry for the covalent binding of Iac azelaic acid to TR at two different p H ' s is presented in Scheme VII. At pH 3.5 two molecules of lac azelaic acid have been bound per active site of TR, whereas at pH 9.5 only one molecule of inhibitor was incorporated. The pH dependence of covalent labelling of the enzyme can be rationalized by the following reaction pathway: (a) Azelaic acid forms an ion pair with the essential base (B) in the active site of TR. (b) The strongly nucleophilic thiolate reacts with the carbonyl-group to form a thioester. (c) The thioester transacylates the base (B) causing enzyme inhibition. (d) The thioester intermediate is stable at pH 3.5 but is readily hydrolysed at pH 9.5, thus explaining the stoichiometry of active site labeling.a9 The inhibition of the reduction of our spin-labeled substrate by azelaic acid at the surface of the skin, at the surface of keratinocytes, melanocytes, melanoma cells and on purified T R ' s from E. coli and human metastatic melanoma membranes, provides imt W O SITES FOR THE INHIBITION OF TYROSINASE BY AZELAIC ACID

uv

~ (." I AZE.

A,C

AC,°

xI04M)

L-TYROSINE

tyrosinase

t'

AZELAIC ACID (K I :2.73 x 10 "$ MI : THIOREOOXIN REDUCTASE (~.

S

• OXIDIZED THIOREDOXIN

(~SH : REDUCEO THIOREDOXIN SH

Scheme VI. The inhibition of tyrosinase by azelaic acid either directly (K~ = 2.73 × 10-~ M) or indirectly on TR (K~ = 12.5 x 10-6 M).

530

K . U . SCHALLREUTERand J. M. WOOD

PROPOSEOMECHANISMFOR THE REACTIONOF 14CAZELAICACIDWITHTR 14COO (C,'H~17 14C00 -

-

BFADH2 SH

"inactive.

pKA SH

.

/

FADH2 l

6.8

.

~.~

-

Active"

.

saoo . / \oH35 "~Acid

"Inactive .

.

.

(883 cpm)

.

Inactive"

(1,624 cpm)

Scheme VII. Proposed mechanism for the inhibition of TR by azelaic acid showing the base-assisted dissociation of active site thiol to nucleophilic thiolate followed by complexation of the azelaic carboxylate group to BH + allowing nucleophilic attack by cys-Sgroup to give an unstable thioester which transacylates B in the active site.

portant confirmation for the specificity for the reaction of nitroxide radical substrate with TR even in the complex biological matrix of the human epidermis. CONCLUSIONS

The TR/thioredoxin electron transfer system has been implicated in a number of reduction reactions in mammalian systems. Its localization in different tissues indicated an important role for these proteins as regulators of the redox balance in the epidermis. Initially it was believed that the TR/thioredoxin system was specific for hydrogen donation to the ribonucleotide reductases. 56 However, seven antioxidant properties for TR have been discovered: (a) Electron donor for the ribonucleotide reductases of bacterial, plant and mammalian systems. (b) The reduction of disulfides, by sulfide interchange reactions, on disulfide proteins such as insulin. (c) The reduction of sulfite to sulfide. (d) The reduction of protein bound methionine sulfoxide back to methionine; an enzyme repair mechanism. (e) The reduction of free radicals. (f) The possible role in the regulation of pigmentation. (g) Membrane bound disulfide reductase in facilitating the transport of re-

duced thioredoxin for filamentous bacteriophage assembly. 56,~7 Primarily TR differs from the other free radical reducing enzymes in its unique location on the plasma membrane, as well as by its presence in the cytosol. 56 Our hypothesis that the TR/thioredoxin system regulates melanin biosynthesis in human skin has been supported by both biochemical and clinical evidence. Competition for electrons between free radicals and thioredoxin has been established with purified TR. 12 Inhibition of tyrosinase by reduced thioredoxin has been proven both kinetically 12and by demonstrating the specific formation of a reduced thioredoxin-tyrosinase inhibitor complex. 16 Experiments with 45Ca+ + indicate that the intracellular equilibrium between oxidized and reduced thioredoxin regulates tyrosinase in tyrosinase positive albinos with HPS. 37 TR activities correlate with pigmentation in the normal healthy adult human population, and activity has been found to be decreased in albinos and in depigmented skin of vitiligo. 13,14The position of the TR/thioredoxin system as both free radical defense system, and in preventing damage to the cell's integrity by hydrogen peroxide, has been confirmed by recent experiments with lens epithelial cells. 59 The authors could show that the TR/thioredoxin electron-transfer reaction was not affected by increasing concentrations of hydrogen peroxide and as little as 9% intracellular reduced thioredoxin was sufficient to protect glyceraldehyde-3-phosphate dehydrogenase activity and stimulated methionine sulfoxide peptide reductase. This property of reduced thioredoxin allowed lens epithelial cells to recover rapidly from hydrogen peroxide toxicity. Very recently TR has been shown to be hyperactive in metastatic melanotic melanoma. 58 These deeply pigmented tumors exhibited TR activities 6-14 fold over normal patient skin type activity. 2° Despite all of the molecular and circumstantial information on the importance of TR in free radical defense and pigmentation, there are still many gaps in our knowledge of this intriguing cellular regulator of redox reactions.

Acknowledgements--We wish to acknowledge the contributions made by our colleagues Dr. Mark R. Pittelkow, Mayo Clinic: Dr. F. K. Gleason, and Dr. Carl Witkop, Jr., University of Minnesota. We wish to dedicate this article to Professor R. J. E Williams, FRS Oxford, for his inspiring work on calcium biochemistry and to Professor Theodor Nasemann, Hamburg for his 65th birthday and in recognition of his service to clinical dermatology.

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K.U. SCHALLREUTERand J. M. WOOD tification of putative heterozygotes. J. Invest. Dermatol. 90:(3):372-377; 1988. Witkop, C. J., Jr. Inherited disorders of pigmentation. In: Goodman, R. M., ed. Clinics in dermatology. Vol. 1. 1985:70-134. Bystryn, J. -C.; Pfeffer, S. Vitiligo and antibodies to melanocytes. In: Bagnara, J. T., ed. Advances in pigment cell research. Vol. 256. New York: Alan Liss; 1988:195-207. Nordlund, J. J.; Abdel-Malek, Z. Mechanisms of post-inflammatory hyperpigmentation and hypopigmentation. In: Bagnara, J. T., ed. Advances in pigment cell research. Vol. 256. New York: Alan Liss; 1988:219-237. Moellmann, G.; Klein-Angerer, C.; Scollay, D. A.; Lerner, A. B. Extracellular granular material and degradation of keratinocytes in the normal pigmented epidermis of patients with vitiligo. J. Invest. Drmatol. 79:321-330; 1982. Bhawan, J.; Bhutani, L. K. Keratinocytes damage in vitiligo. J. Cutan. Pathol. 10:207-212; 1983. O'Donnell, M. E.; Williams, C. H., Jr. Reactions of both active site thiols of reduced thioredoxin reductase with N-ethylmaleimide. Biochemistry 24:7607-7621; 1985. Nazzaro-Porro, M.; Passi, S. Identification of tyrosinase inhibitors in cultures of pityrosporium. J. Invest. Dermatol. 71:205208; 1978. Nazzaro-Porro, M.; Passi, S.; Balus, L.; Breathnach, A. S. Effect of dicarboxylic acids on lentigo maligna. J. Invest. Dermatol. 72:296-305; 1979. McLean, D. 1.; Peter, K. K. Apparent progression of lentigo maligna to invasive melanoma during treatment with topical azelaic acid. Brit. J. Dermatol. 114:685-689; 1986.

51. Doherty, V. R.; Ashworth, J.; Cox, N. Azelaic acid in lentigo maligna. British J. Dermatol. 116:606; 1987. 52. Nazzaro-Porro, M.; Passi, S. Inhibition of tyrosinase by azelaic acid. Pigment Cell 4:234-243; 1979. 53. Schallreuter, K. U.; Wood, J. M. Kinetics for the inhibition of tyrosinase by azelaic acid. British J. Dermatol. in press; 1988. 54. Breathnach, A. S.; Ward, B. J.; Robins, E. J.; Bashim, Y.; Ethridge, L.; Passi, S,; Nazzaro-Porro, M. Analytic ultrastructural autoradiographic localization by 3H dicarboxylic acids in cultured normal human melanocytes and keratinocytes. In: Bagnara, J. T.; Klaus, S. N.; Paul, E.; Schartl, M., eds. Biological, molecular and clinical aspects of pigmentation. XIII'h International Pigment Cell Conference; 1985:627-632. 55. Leibl, H.; Stingl, G.; Pehamburger, H.; Korschan, H.; Konrad, K.; Wolf, K. Inhibition of DNA synthesis of melanoma cells by Azelaic acid. J. Invest. Dermatol. 85:417-422; 1985. 56. Holmgren, A. Thioredoxin. Ann. Rev. Biochem. 239:149-180; 1985. 57. Russel, M.; Model, P. The role of thioredoxin in filamentous phage assembly. Proc. Nat. Acad. Sci, USA 82:29-33; 1985. 58. Schallreuter, K. U.; Wood, J. M.; Breitbart, E. W.; Kimmig, W.; Hicks, R.; Radloff, H.; Janner, M. Thioredoxin reductase in primary melanoma and surrounding skin. Eur. Soc. of Pigment Cell Research, First meeting. Sorrento, Italy. Proceedings abstr. 95:41; 1987. 59. Spector, A.; Yan Guo-Zai, Y.; Huang Ruey-Ruey, C.; McDermott, M. J.; Gascoyne, P. R. C.; Pigiet, V. The Effect of H202 upon thioredoxin-enriched lens epithelial cells. J. Biol. Chem. 263(10):4984-4990; 1988.