The Journal of Pathology

Vol. 97 No. 2

HYDROXYANISOLE DEPEGMENTATION: IN-VIVO STUDIES

P. A. RII,EY * Department of Chemical Pathology, University College Hospital Medical School,

University Street, London

PLATES LXXVIII-LXXXI

SINCE the original reports of Oliver, Schwartz and Warren (1939,1940) describ- ing the depigmenting effect of the monobenzyl ether of hydroquinone, a number of studies have been made to elucidate the mechanism of action of this and allied substances on melanogenesis (Lorincz, 1950; Lea, 1951 ; Denton, Lerner and Fitzpatrick, 1952).

Considerable attention has been focused on the antioxidant properties of hydroquinone and its derivatives, and in an extensive survey of antioxidants Brun (1961) found that para- hydroxyanisole had very powerful depigmenting activity. The descriptions of chemically induced depigmentation suggested similarities to the depigmentation occurring in vitiligo.

The present study was therefore undertaken to investigate (I) the extent of the similarity between para (p)-hydroxyanisole depigmentation and vitiligo, and (2) the degree of chemical specificity requisite for the depigmenting action.

MATERIALS AND METHODS

The experiments were made on pure black guinea-pigs bred from stock originally obtained from Animal Supplies, London, and fed on MRC Diet SG1 and water ad libitum.

Materials. The agents employed were as follows : p-hydroxyanisole (Koch-Light Ltd), meta (m)-hydroxyanisole (redistilled from material supplied by Eastman Kodak), ortho (0)- hydroxyanisole (Eastman Kodak), 0-, m- and p-fluoroanisole, and the corresponding isomers of chloroanisole and bromoanisole (Koch-Light Ltd), p-hydroxydiphenyl (Aldrich Inc.), p-hydroxybenzanisole (Koch-Light Ltd), L-tyrosine (Hopkin and Williams Ltd), 3-chloro- tyrosine, p-fluorophenylalanine, p-chlorophenylalanine (Koch-Light Ltd), p-hydroxyphenyl- pyruvate (British Drug Houses Ltd), adrenaline, noradrenaline, 3-methoxynoradrenaline, dihydroxymandelic acid and 3-methoxymandelic acid (Sigma Chemical Co. Inc.).

Sodium ascorbate was prepared from ascorbic acid (Hopkin and Williams Ltd). D-E-

tocopheryl polyethylene glycol 1000 succinate was obtained from Distillation Products Industries.

Animals.

Received 10 May 1968; accepted 24 June 1968. * Beit Memorial Research Fellow.

J . PATH.-VOL. 97 (1969) 185 N

186 P. A . RILEY

Application. In preliminary tests it was found that the animals would not accept drinking water containing concentrations of hydroxyanisole above 0.1 per cent., and, although they would eat a blended mixture of food andp-hydroxyanisole crystals in proportions of 70 : 30, there was no evidence of an effect on pigmentation. Local application of p-hydroxyanisole, however, was found to have a marked depigmenting effect and local applications were there- fore employed throughout this study. The test substances were made up in molar ratios comparable with a 20 per cent. cream of hydroxyanisole in lanolin (70 per cent. anhydrous wool fat, 29 per cent. water, 1 per cent. cetyl alcohol). Applications were made once daily to the back of the ears for periods between 1 and 8 wk. Control animals were treated with lanolin alone.

Assessment of depigmenting action. The effects on pigmentation were assessed visually and samples of treated skin for further examination were obtained by biopsy under general anaesthesia (halothane and oxygen) from the back of the ears and from the hairless area behind the ears. Thin slices were cut freehand with a dermatome knife and processed as follows. Material for cryostat sections was: either fixed for 30-60 min. in ice-cold 4 per cent. formalin in 0 . 0 5 ~ barbital buffer (pH 7.2) containing 1 per cent. calcium chloride, washed in distilled water and mounted in 5 per cent. aqueous carboxymethyl cellulose; or mounted directly in carboxymethyl cellulose on a chuck in the manner described by Cane (1967). Sheets of epidermis were obtained by immersing the biopsy material in 2~-CaC12 at room temperature for 20 min. : the dermis could then be gently detached with fine forceps under a dissecting microscope. The detached epidermis was fixed in ice-cold buffered formol- calcium. The presence of tyrosinase activity was determined by incubating fixed material for 2 hr at 37°C in 0.005~ dihydroxyphenylalanine made up in 0 . 0 1 ~ phosphate buffer (PH 7.1). Nucleoside polyphosphatase activity was demonstrated by the calcium-cobalt method for adenosine triphosphatase (ATPase) (Padykula and Herman, 1955).

For the grafting experiments Thiersch grafts were taken from the backs of the ears of adult female guinea-pigs under general anaesthesia by means of a small dermatome designed to enable a standard 75 mm2 area of skin to be removed (Bowler and Riley, 1968). Auto- grafts were made by reciprocal transfer of the skin removed from the left ear to the right ear and vice versa. The grafts were kept in place with tulle gras and the ears clipped together to prevent the animals from scratching the grafts.

Skin penetration. The keratin surface of full thickness guinea-pig skin taken from the back of the ears by means of the microdermatome was glued to the end of lOcm lengths of glass tubing with Bostik no. 1 adhesive applied to the rim of the tube to form a seal over the end. The internal diameter of the tubing was 3.5 mm, so that 0.096 cm2 of skin formed a membrane through which the methoxyphenol was required to penetrate. Each tube was immersed in 2.5 ml Krebs-Ringer-bicarbonate containing 200 mg glucose per 100 ml and tested for leaks. After addition to the tubes of 0.1 ml of 5 X 10-2M-hydroxyanisole solution in neutralised distilled water, the tube height was adjusted so that the menisci of the fluids inside and outside the tubes were level and the tubes were clamped. The rate of penetration was estimated by spectrophotometric readings of the amount of hydroxyanisole in the Krebs- Ringer-solution at 10-min. intervals. It was found that the initial rate of penetration became more rapid with ageing of the system and a correction curve was made for each membrane sample based on repeated readings of the initial penetration rate of p-hydroxyanisole.

RESULTS Visual assessment of in-vivo eflects of locally applied agents

It was found that complete depigmentation occurred with applications of 20 per cent. p-hydroxyanisole, starting within 5-10 days of the beginning of treatment. The depigmentation extended over the region of application with further treatment. After 5-6 wk large areas appeared completely depigmented (fig. la). The rate of depigmentation was found to be more rapid in young

HYDROXYANISOLE DEPIGMENTATION 187

guinea-pigs than in adults, in which up to 8 weeks’ treatment was necessary to produce totally depigmented regions of skin of comparable size. In none of the 34 animals treated with p-hydroxyanisole did the depigmentation extend beyond the area of application. On cessation of treatment the totally depig- mented regions became repigmented by pigment spread from the margins and from small perifollicular zones of pigmentation (fig. lb). This repigmentation took place very slowly in the hairless areas and many zones of depigmentation persisted for over 6 mth.

TABLE I Depigmenting action of locally applied agents

Compound

o-H y droxyanisole rn-Hy droxyanisole p-Hydroxyanisole p-Fluoroanisole p-Chloroanisole p-Bromoanisole p-Hydroxydiphenyl p-Hydroxybenzanisole L-Tyrosine 3-Chlorotyrosine p-Fluorophenylalanine p-Chlorophenylalanine p-Hydroxyphenyl-

pyruvate Adrenaline Noradrenaline Methoxynoradrenaline Dihydroxymandelic acid 3-Methoxymandelic acic

-0CH3

-0CH3

-0CHx

-OCH3

-OCH3

-0CH; -C6HS

-OCHzC6H5 -CH2 .CHNHz. COOH -CH7 .CHNH> .COOH -CH;.CHNH;.COOH -CH2. CHNHr .COOH

-CH2. CO. COOH

-CHOH . CH2. NHCH3 -CHOH. CH2NHz -CHOH . CHzNHz -CHOH . COOH -CHOH . COOH

-OH ... ... ... ... ... ... ... ... ... ... ... ...

...

...

...

...

...

R3*

... -OH ... ... ... ... ... ... ... -c1 ... ... ...

-OH -OH

-OH -0CH3

-OCH3

R4*

...

... -OH -F -C1 -Br

-OH -OH -OH -OH -F -c1

-OH

-OH -0 H -OH

-OH -OH

* The radicals are given where they differ from -H.

In some animals treated with rn-hydroxyanisole a noticeable lightening of the treated skin was observed and small zones of depigmentation were observed. The effect was, however, much less marked and took longer (4-5 wk) to develop than with p-hydroxyanisole. Animals treated with o-hydroxyanisole or with lanolin alone did not show any change from the untreated state. Similarly no depigmentation was produced by local application of other agents with the exception of p-hydroxybenzanisole (table I).

Combined applications of p-hydroxyanisole with other agents Antioxidants. Combinations ofp-hydroxyanisole with 5-40 per cent. sodium

ascorbate and with the equivalent of 5 per cent. cr-tocopherol as D-cr-tocopheryl

188 P. A. RILEY

polyethylene glycol 1000 succinate were also employed in order to test their effect on p-hydroxyanisole depigmentation. Ascorbate had no effect on the rate or extent of p-hydroxyanisole-induced depigmentation. Vitamin E lengthened the time before depigmentation became evident to between 12 and 16 days.

Tyrosine. Tyrosine applications led to increased local pigmentation, and in addition considerable hair loss was observed in the treatment area. Corn- binations of tyrosine with p-hydroxyanisole in molar proportions of 1 : 1 and 1 : 10 resulted in complete depigmentation, as with p-hydroxyanisole alone. If the proportions were 10 : 1, depigmentation was observed after 4 wk.

Grafts Seven of 8 grafts were successful. In 2 animals the transferred autografts

were between a p-hydroxyanisole-treated depigmented ear and an untreated pigmented ear. In these the pigmented skin in the unpigmented host site remained pigmented and in one case pigment-spread into the surrounding skin took place (fig. 2). The depigmented skin transferred to the normal sites slowly repigmented, in both cases apparently from endogenous islands of pigment cells. The remaining 2 animals had the grafts exchanged between p-hydroxyanisole- treated depigmented sites. In these the repigmentation was more rapid than in the surrounding skin and was complete within 4 wk of the time of grafting, which was performed 1 wk after the cessation of treatment.

Light microscopy Examination of vertical sections of skin treated with hydroxyanisole confirms

the macroscopic appearances in that pigment granules in the epidermal cells become progressively more scarce as treatment with p-hydroxyanisole proceeds. Control sections and sections of skin from animals treated with other agents (except m-hydroxyanisole) are heavily pigmented throughout. A slight increase in epidermal thickness is noted in all the hydroxyanisole-treated samples during the period of treatment and in the case of p-hydroxyanisole some exaggeration of the granular layer is noted. In other respects the skin appears normal.

Dopa reaction. In p-hydroxyanisole-treated skin a progressive reduction in the numbers of dopa oxidase (tyrosinase) positive epidermal dendritic cells begins about 4 days after treatment commences. Separated epidermal sheets examined during the first 10 days’ treatment with p-hydroxyanisole after incubation in dopa show two morphologically distinct types of cell (fig. 3 ) : one type showing only weak tyrosinase activity and clearly dendritic in shape; and another type densely stained with dopa oxidation product, but small and irregular in shape with few or no dendrites. Many of this latter type of cell are located above the basal layer, whereas the branched cells are always basally situated. At later stages in the treatment the appearances are different. Most regions fail to show any cells with dopa oxidation product and those that remain are basally sited in relation to hair follicles, are dendritic in shape and stain densely with tyrosinase reaction product (fig. 4). Because of the patchy nature

RILEY PLATE LXXVIII

HYDROXYANISOLE DEPIGMENTATION

FIG. la.-Skin during treatment with F k . 1b.-Skin 5 wk after cessation of p-hydroxyanisole. treatment. Extending zones of repigmen-

tation. Note depigmented hair at margins of treated area, and new growth of pig- mented hair.

FIG. 1 .-Experimentally depigmented region. x 3.2.

FIG. 2.-Site of a pigmented graft made into a depigmented region, showing extent of pigment spread into surrounding skin after 3 wk. Graft margins indicated by interrupted lines. x 3.

RILEY PLATE LXXIX

HYDROXYANISOLE DE:PIGMENTATION

FIG. 3u.--General view, showing (arrows) FIG. 3b.-Basal branched cell and (arrow) both weakly stained dendritic cells and aniorphom cell at different plane of focus. heavily stained amorphous cells. x 575. x 850.

FIG. 3.-Froni an animal treated for 7 days with p-hydroxyanisole. Separate ear epidermis incubated to show tyrosinase activity.

FIG. 4u.-Dopa-positive melan- FIG. 4b.--Dopa-positive melanocytes in a repigmenting ocytes in a hair follicle. x 320. region surrounding a hair follicle. x 450.

FIG. 4.-Puva-hydroxyanisole-treated guinea-pig. Epidermis incubated in dihydroxyphenylalanine medi um.

HYDROXYANISOLE DEPIGMENTATION 189

o-Hydroxyanisole m-Hydroxyanisoie p-Hydroxyanisole Untreated

of the effect on the distribution of dopa-oxidising dendritic cells a quantitative assessment cannot usefully be made. Qualitatively there is a progressive reduction in the numbers of tyrosinase-positive cells. The last regions to be affected in this way are the perifollicular zones of ear skin. Tyrosinase-positive cells are absent in depigmented skin removed up to 3 mth after treatment has been stopped (fig. 5). The small depigmented regions of rn-hydroxyanisole- treated skin also show areas in which the dopa-oxidase reaction is absent.

Skin from o-hydroxyanisole- and lanolin-treated animals shows no reduction in either the numbers of or density of reaction product in melanocytes compared with material from untreated sites. No change is noted in the dopa-oxidase reaction of dendritic cells in the skin of animals receiving tyrosine or the other agents.

ATPase activity. Epidermal dendritic cells manifesting ATPase activity are present in all material examined. Cell counts made on vertical sections of hydroxyanisole-treated skin after three weeks’ treatment give higher values in epidermis depigmented by p-hydroxyanisole than in normal or tyrosine-treated pigmented skin or in pigmented skin after treatment with 0- and rn-hydroxy- anisole (table I1 and fig. 6). The reaction product appears more dense in the depigmented epidermis, but this may be due to greater contrast in the absence of pigment.

TABLE 11

Counts of suprabasal epidermal dendritic cells in ear skin from guinea-pigs treated locally with hydroxyanisoles

4.1 0.08 4 4.5 045 6 5.9 0.35 15 4.4 0.23 4

Mean cell ~~~~~ Number of 1 number 1 mean I samples Compound

The cells were demonstrated by their ATPase activity and counted with an eyepiece graticule. The mean values are calculated from the average counts of several samples and expressed as cells per 70,000 nm3fstandard error.

Separated epidermal sheet preparations from both pigmented and depig- mented skin show a similar distribution of basal ATPase-positive dendritic cells

Comparative rates of penetrat ion of hydroxyanisoles

The rate of penetration during the initial ten minutes’ exposure to an aqueous solution of the agent was found to be similar for 0-, m- andp-hydroxy- anisole. The mean initial absorption rates for three separate skin samples were (10-4 moles per cm2 per min.): for the ortho isomer, 4.71 ; meta, 9.41; para, 8-05.

(fig. 5).

190 P. A . RILEY

DISCUSSION Similarity of experimentally produced depigmentation to vitiligo

The results of the present study indicate that the depigmentation produced by local application of hydroxyanisole corresponds to vitiliginous depigment- ation in the following respects: (1) the irregular and extending nature of the loss of pigment during treatment; (2) the tendency for hair follicles to depigment last ; (3) repigmentation from remaining perifollicular melanogenic cells and by pigment spread from the margins of depigmented areas during recovery (Pegum, 1955); (4) the behaviour of the grafts (Haxthausen, 1947-48; Spencer and Tolmach, 1952; Kato, 1955); (5) dopa-oxidising epidermal dendritic cells were absent or reduced in number in the depigmented zones as in vitiligo (Jarrett and Szabo, 1956); (6) basally situated epidermal dendritic cells demon- strating ATPase activity were present in the depigmented skin (Brown, Winkle- mann and Wolff, 1967; Riley, 1967); and (7) the suprabasal ATPase-positive dendritic cells were increased in numbers in the depigmented regions as has been reported for vitiligo (Riley, 1967). The evidence from all these aspects points to a fundamental similarity between vitiligo and the experimental system presented by p-hydroxyanisole depigmentation.

Chemical speciJicity of depigmenting agent The data summarised in table I indicate that considerable specificity exists

in the type of substance that will cause depigmentation since minor alterations in structure lead to considerable differences in effectiveness.

The basic requirements are a meta or para ring hydroxyl group, with markedly greater effectiveness in the para position, and a non-polar side chain possibly requiring an ether linkage. It is not certain to what extent the side chain might alter the degree of penetration of the agents tested, but the differential effectiveness of hydroxyanisole with ortho-, meta- or para-positioned hydroxyl groups in causing depigmentation cannot be ascribed to differ- ences in the penetration of these substances, since the absorption rates were shown to be closely similar.

Nature of depigmenting action The nature of the mechanism of p-hydroxyanisole depigmentation is not

It is convenient to consider the possible mechanisms as having either primary or secondary effects on pigment production. It is worth while, in considering primary effects, to remark on the similarity of the structure of p-hydroxyanisole to tyrosine. This might suggest com- petitive inhibition of tyrosinase as the mode of action of substituted phenols, but this is excluded by the negative dopa reaction in the depigmented zones. Since the incubation is made in the presence of a large excess of dihydroxyphenylalanine it is unlikely that this substrate would fail to be oxidised by virtue of competition for the active site of tyrosinase by any small residual amounts of p-hydroxyanisole in the tissue and, moreover, the dopa reaction remained negative in some areas of depigmented skin for many weeks after the cessation of treatment with p-hydroxyanisole. In addition there is evidence that the in-vitro effect of p-hydroxyanisole is to stimulate tyrosine oxidation (Riley, 1969).

known.

HYDROXYANISOLE DEPIGMENTATION 191

Alternatively it is possible that p-hydroxyanisole exerts its depigmenting effect by blocking the synthesis of tyrosinase in melanocytes. If it were assumed that the genetic regulation of tyrosinase synthesis, at either the transcriptional or the translational level, is brought about by a system in which the substrate for the enzyme acts as the inducer, then tyrosinase syn- thesis could be inhibited by a tyrosine analogue that was able to bind to the inducer site, but failed to have an inducing effect. Consistent with this is the increased pigmentation produced by local application of tyrosine, and a 10 : 1 excess of tyrosine overp-hydroxyanisole delayed depigmentation; the ratio of tyrosine to p-hydroxyanisole would presumably indicate the inverse ratio of their binding constants to the hypothetical repressor. There is, however, no direct evidence at present to suggest that tyrosine acts as an inducer of tyrosinase synthesis in melanocytes.

One is led, therefore, to conclude that g-hydroxyanisole has a cytotoxic effect that secondarily interferes with the capacity of melanogenic cells to synthesise pigment.

SUMMARY A vitiligo-like depigmentation is produced by the topical application of

This depigmenting action is confined to three closely related compounds

The significance of this chemical specificity is discussed in relation to the

para (p)-hydroxyanisole in vivo.

within a range of analogous substances tested.

possible mode of action of p-hydroxyanisole.

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