Role of Thiol Compounds in Mammalian Melanin Pigmentation: Part I. Reduced and Oxidized Glutathione
Journal of Investigative Dermatology
Evidence for the postulated role of glutathione reductase in melanin pigmentation has been obtained by determinations of the glutathione concentrations in Tortoiseshell guinea pig skin of differe nt colors (black, yellow, red, and white). As expected, the lowest levels of reduced glutathione (GSH) were found associated with eumelanin type pigmentation, whereas the highest ones were found in the skin with phaeomelanin producing melanocytes. On the other hand, white skin of guinea pig having no
... nea pig having no active melanocytes showed GSH levels which were intermediate between those of the black and yellow areas. These results are consistent with the view that the activity of the enzyme glutathione reductase, though not primarily related to pigmentation, plays an important role in the regulation and control of the biosynthetic activity of melanocytes leading to various types of melanin pigments_ DUl'ing the 1940's Flesch and Rothman [1,2] re ported exp erimental evidence suggesting that sulfhydryl compounds, such as glutathione and cysteine, are capable of inhibiting t h e biosyn t h esis of m ela nin by combining with the copper present in tyrosinase. Since t h e n t his view has b een widely accepted as a regulatory m ech a nism in m elanin pigmentation r eceiving a dditional support thTough subsequent studies by Halprin and Ohkawara [3, 4] . These investigators s howed that the con centration of reduced glutathione in huma n s kin is som e 100 t imes the minimum con centration n ecessary to observe inhibition of m ela nin form ation. They also showed that N egroid skin contains less redu ced glutathione and glutathione reductase than does Caucasoid skin. However, as our knowledge of the ch emistry of melanogenesis h as improved in recent years [ 5, 6] , it has becom e increasingly clear t h at sulfhydryl compounds have little or no effect on the catalytic activity of tyrosinase but t h ey act as ch e mical scavengers for dopaq uinone form ed within m ela nocytes to give . colorless adducts, e.g., the cysteinyldopas [7, 8] . These may be either oxidized wit h production of yellow or reddish-brown phaeo m e lanins, or take part in the biosynthes is of certa in Manuscript This work was supported in pal-t by '1 grant from "U.E. R Biologie Humaine-Faculte de Medec in e" Lyon-Jean-Pierre Benedetto is a rec ipi ent of a sc holarship from Institut National de la Sante et la Recherche Medicale. (INSERN) Reprint requ ests to: J . Thivolet, U.209 " Rec herche dermato logique et immunologie," Hopita l Edouard Herriot, 69374 Lyon eedex 2, France. Abbreviatio ns: DTNB: 5,5'-dithiobis (2 nitrobe nzo ic ac id) EDTA: ethylene diamine tetracetate GS H: reduced glu tathione GSSG: oxidized glu tathione HP03: metaphosphoric ac id s ulfur-containing eumelanins [9,10), or r eleased from m ela nocytes to be eventua lly excreted in the urine    . Though these facts are well known, no atte mpt has been made in r ecent years to correlate the sulfhydryl conte n t of t h e s kin with the type of pigm entation. We h ave the refor e b egun s u ch a study, and our preliminal'Y observations on t h e glutathione levels in guinea pig skin of different colors co nstitute t h e su bject of this r e port. MATERIALS AND METHODS Tortoiseshell guinea pigs e"e" with whi te spotting [1 4] show ing a 3 co lored coat were studied. Two different groups of animals we re examin ed: one showing a mixture of white, black, and red areas; the other with white, black, and ye llow patches. These anima ls were obtained from the breeding la borato ry of Institut Nationa l des Sciences Appliques de Lyon (Service du Professeur Laviolette). Reduced (GSH) a nd oxidized (GSSG) glu tathione, N ethylmaleimide O. phtalaldehyde were obtained from S igma chemical co mpagny (St. Louis, Mis.). All determin ations of fluorescence inte nsity were perform ed wit h a !luorocolor _ imeter Aminco. Tissue Prepara.tion Guinea pigs were killed by a blow on head then sheaved. The skin was removed, used immediate ly or frozen and kept at -80 0 C until use. 150-250 mg of tissue was homogenized at 2_3° e using a polytron homogenizer in a phosphate buffer, 0,1 M, EDTA, 5 mM; pH 8.0. 4% HPO" was used as a protein precipitant. The homogena te was ce ntrifuged (3 x 10 H g min at 4°e ). GSH and GSSG were measured in t he supernatant. Light Mi croscopy For light mi croscopy solubili ty test, skin biopsies were frozen with li quid nitrogen. Sections (8 J.Lm thi ck) were cut on a cryoc ut. Solu bility tests were carri ed out under a ligh t microsco pe by treating sections in a sodium hydroxide solu tion 0.25 M  . 402 Split DOPA Studies Biopsies of the skin of different colors (red, ye llow, black, white ) were incubated in 2 N NaB I' solution at 37°e for 45 min and t he separated epidermis and hair follicles t hus obta ined were incubated with 0.1% DOPA so lutions in 0.1 sodium phosphate buffer (pH 7.4) for 4 hI' at 37°e. After the dopa reaction, the tissues we re fixed with 10% neutral form alin, dehydrated in alcoho l and moun ted. Electron. Micro scopy S kin biopsies of the different colors were fixed in 3% glutaraldehyd e for 1 hI', postfixed in osm ic acid for 1 hI', dehydra ted in alcohol, embedded in Epon and sectioned on a Reichert OM U3 ultram icrotome. The sections were stained with uranyl acetate and lead citrate and examined with an Hi tachi H U 12 A elec tron microscope. Glutathione A ssays Bot h redu ced and oxidized glu tathione (GSH and GSSG) we re measured by a specific and very sensitive nu orometric method desc ribed by Co hn and Lyle [1.6] and later modified by Hissin anel Hilf (1 71, This method takes advantage of the reaction of ortho ph talaldehyd e at pH 8.0 with GSH and at pH 12.0 with GSSG. N ethylmaleimide is used to preve nt in terference ofGSH with th e measurement ofGSSG.