MECHANISM OF PIGMENT-CELL TOXICITY P R O D U C E D BY HYDROXYANISOLE

P. A. RILEY* Department of Chemical Pathology, University College Hospital Medical School, London

PLATES LXVIII-LXXI

RILEY (1969a and b) presented evidence indicating a preferential cytotoxic effect of certain substituted phenols, notably 4-hydroxyanisole, on melanocytes. It was suggested that a possible mechanism of this selective toxicity was the oxidation of these substances by tyrosinase to give rise to free radical derivatives that could initiate lipid peroxidation and consequent cell damage (Slater and Riley, 1966). In the present paper further evidence is presented that supports this view of the cytotoxic action of hydroxyanisole and other phenols.

MATERIALS AND METHODS

In-vitro experiments were carried out on primary cultures of normal guinea-pig melano- cytes obtained at biopsy under halothane anaesthesia from an inbred strain of black guinea- pigs fed on MRC diet 41, and water ad libitum. The same stock animals were also used for in-vivo experiments. The cells were cultured by a procedure modified from that of Prunikras, Leung and Colson (1964), and grown in Cruickshank chambers (Cruickshank, Cooper and Conran, 1959) in Hanks’ BME with 5 per cent. foetal calf serum containing penicillin and streptomycin (100 units per ml) and 0.1 per cent. chick embryo extract. These materials were obtained from Flow Laboratories Ltd, Irvine, Scotland. Cultures of melanocytes showing DNA, RNA and protein synthesis were obtained. The details of the method and the behaviour of these cells in culture are published elsewhere (Riley, 1969~). Cultures between 4 and 28 days old were used. The procedure adopted was to introduce medium containing the agent to be tested into the chamber. Changes were then observed by direct observation and by time-lapse cinematography. For some experiments the chambers were washed out with standard medium four times after a timed exposure of the cells to the agent. In other instances the cells were fixed at the end of exposure with 2 per cent. glutaraldehyde in 0 . 1 ~ phosphate buffer (PH 7.4) for 30 min. at 4°C.

Autoradiography. The uptake of 4-hydroxyanisole by melanocytes was investigated by using radioactive 4-hydroxyanisole labelled by equilibration with tritiated water to a specific activity of 372 mCi per m.mole. This material was prepared by the Radiochemical Centre, Amersham, and purchased with a special grant from the Beit Memorial Fellowship Trustees. The material was introduced into the chambers in concentrations of 0-2 mCi per ml for periods of 30 min., 12 and 24 hr. The cells were fixed and the coverslips removed and coated with Ilford K2 Nuclear Emulsion in gel form by a modification of the technique of Kopriva and Leblond (1962). Exposure time was 3 wk. Fixed material was stained with haematoxylin, dehydrated, cleared in xylol and mounted in Zeiss mounting medium.

Dopa reaction. The distribution of tyrosinase activity was investigated by incubating the fixed cells in 0 . 0 5 ~ L-3,Cdihydroxyalanine in 0.1~ phosphate buffer at p H 7.0 for 2 hr.

Electron spin resonance. In-vivo experiments were made to establish whether the local

Received 14 Nov. 1969; accepted 9 Jan. 1970. * Wellcome Research Fellow.

J. PATH.-VOL. 101 (1970) 163

164 P. A. RILEY

application of a cream containing 20 per cent. 4-hydroxyanisole would produce a detectable alteration in the electron spin resonance signal of black guinea-pig ear skin. Samples from guinea-pigs treated for 8 days and from untreated control animals were taken by Tiersch biopsy and frozen immediately in liquid nitrogen. The frozen samples were ground up by hand with a pestle and the powder placed in a 3-mm-diameter Spectrosil A Quartz sample tube to a height of 8 mm. Readings were made on a Decca X-1 electron spin resonance spectro- meter operating at X band (9270 MHz) with simple detection and 100 kHz field modulation. The magnet system was Newport Instrument type M2X with automatic field readout. The sample was kept cold in a Decca Variable temperature cavity insert and examined at - 180°C. Recordings were made with a time constant of 1 s, modulation - 10 db, scan 20 gauss (G) per in. and sweep time of 500 s.

RESULTS Autoradiographs

Autoradiographs of mixed melanocyte and keratinocyte cultures show a selective uptake of 3H-4-hydroxyanisole by pigment cells (figs. 1-4). When the labelled compound is administered for periods exceeding 12 hr there is heavy labelling of clumps of pigment granules present in keratinocytes (fig. 4), whereas after shorter periods of incubation no such labelling is detected, which suggests that the labelled pigment granules were transferred from melanocytes in the intervening period. In melanocytes the labelling is associated with regions of the cytoplasm containing melanosomes and is sparse over regions of cytoplasm devoid of melanosomes (fig. 3). Attempts were made to define more precisely the site of labelling by electron-microscope autoradiography, but this approach was invalidated by the diffusion of labelled material into the Araldite during processing, although heavy labelling overlying melanosomes is found. The material diffusing into the resin may have been adsorbed on the cell surfaces (Riley and Seal, unpublished).

Dopa reaction In general there is a progressive loss of pigmentation of melanocytes during

culture, matched by a reduction in the number of cells displaying a positive dopa reaction. The reaction product is found to be closely associated with the distribution of melanosomes in melanocytes. In mixed cultures there is rarely any reaction product associated with melanin granules present in keratinocytes. Comparison of cells in cultures of equivalent age and state of pigmentation processed to show tyrosinase activity and labelled with 4-hydroxyanisole shows a correspondence of distribution of dopa reaction product and autoradio- graphic grains.

EfSect of hydroxyanisoles The three structural isomers of hydroxyanisole were tested in final con-

centrations between 5 x 10-3 and 10-10 M. At 10-3 M 4-hydroxyanisole is extremely toxic to melanocytes, but similar amounts of 3-hydroxyanisole and 2-hydroxyanisole are without effect as judged morphologically after a period of 30 minutes’ exposure (figs. 5-11). The time-course of events in the case of 4-hydroxyanisole intoxication is as follows. No effect is observed for about

RILEY PLATE LXVIII

HYDROXYANISOLE AND PIGMENT-CELL TOXICITY

FIG. I .-Note grains overlying pigment in the dendritic processes.

FIG. 2.-Relative absence of grains in the cytoplasm over the nucleus.

FIGS. 1 and 2.-Autoradiographs of cells labelled in vitro with tritiated Chydroxyanisole showing that the distribution of grains corresponds to the distribution of pigment granules. x 680.

FIG. 3.-Note the sparing of two unpigmented FIG. 4.-Note dense grains overlying pigment package in keratinocyte cytoplasm. zones near the nucleus.

FIGS. 3 and 4.-4-Hydroxyanisole uptake. Autoradiographs. X 1400.

RILEY PLATE LXlX

HYDROXYANISOLE AND PIGMENT-CELL TOXICITY

FIG. 5.-Effect of l o - 3 ~ l-tert-butyl- FIG. 6.-Effect of 30 minutes’ exposure catechol. Note severe effect on heavily to l o - 3 ~ 4-hydroxyanisole. Note pigmented melanocyte after 30 minutes’ that the keratinocyte is unaffected. exposure. x 800. x 473.

FIG. 7.-Early toxic changes in pigmented but not in unpigmented melanocytes after 10 minutes’ exposure to l o - 3 ~ 4-hydroxyanisole. Phase-contrast. x 608.

RILEY PLATE LXX

HYDROXYANISOLE AND PIGMENT-CELL TOXICITY

Fiti. 8.-Cytoplasmic bIebs forming on a melanocyte exposed to 1 0 - 3 ~ Chydroxyanisole for 5 min. Phase-contrast . x 1700.

FIG. 9.-Disruption of melanocyte after 15 minutes’ exposure to 10- 3~ 4-hydroxyanisole. Phase- contrast. x 1550.

RILEY PLATE LXXI HYDROXVANISOLE AND PIGMENT-CELL TOXICITY

FIG. 10.-Melanocyte appearance after 12 hours’ exposure to 1 0 - 5 ~ 4-hydroxy- anisole. Note the pyknotic nucleus. ‘x 600.

FIG. 11 .-Cells unaffected by l o - 3 ~ 3-hydroxy- anisole for 12 hr. X 600.

FIG. 12.-Partial protection to 30 minutes’ exposure FIG. 13.-Effect of ubiquinone in the presence to 10-3~ 4-hydroxyanisole produced by diethyl- of l o - 3 ~ 4-hydroxyanisole. One cell has been dithiocarbamate. X 600. disrupted after 20 minutes’ exposure, whilst

others have been protected. X 600.

HYDROXYANISOLE AND PIGMENT-CELL TOXICITY 165

5 min., after which blebs of thin cytoplasm appear to extrude from random points over the surface of melanocytes (fig. 8). These changes resemble those described in phototoxic damage to cultured cells (Allison, Magnus and Young, 1966). In some instances the cell surface ruptures and cytoplasmic granules spill out into the medium (fig. 9). After 15-30 minutes’ incubation in the

TABLE I Efect of varying concentrations and exposure times on damage to melanocytes

treated with 4-hydroxyanisole

Concentration of 4-hydroxyanisole (M)

5x 1 0 - 3

1 x 10-3

1 x 10-4 5 x 1 0 - 5

4 x 10-6

1 x 10-6

1x10-8

5x 10-9

1 x 10-10

Time of exposure

3 min. 15 min. 30 min.

30 min. 30 min. 30 min.

30 min. 30 min.

30 min. 12 hr

60 min.

30 min. 60 min. 12 hr 24 hr 36 hr

24 hr

12 hr 24 hr 36 hr

Damage to melanocytes

- -

+++ +++ +++ +++ ++ + + - -* - - - + ++ + - - -

* Electron micrographs of cultures treated with this dilution of 4-hydroxyanisole for 30-60 min. showed cells denuded of cell membrane.

presence of 4-hydroxyanisole all the melanocytes in young cultures show gross morphological evidence of intoxication. In cultures 10-14 days old in which pig- mentation and the dopa reaction are much reduced, the evidence of toxicity is greatly diminished and frequently absent (fig. 7). Keratinocytes present in mixed cultures show no progressive toxic changes (fig. 6) although initial effects are observed, such as the production of “ microspikes ” or filiform pro- cesses and cortical budding. These are described in detail elsewhere (Riley and Seal, unpublished).

4-Hydroxyanisole has a melanocytotoxic effect in dilutions down to 10 -8 M, although progressively longer incubation periods were required to reveal this (table I). Time-lapse cinematography shows that concentrations of 10 -6 M cause an initial increase in the rate of movement of cells and the breakage of

166 P. A. RILEY

Agent

previously stable cell contacts. No effects are observed after 36 hours’ exposure to 4-hydroxyanisole at concentrations of 10 -10 M.

Other substances that were found to be toxic to cultured melanocytes in concentrations of 10 -3 M were: isopropyl catechol, tert-butyl catechol, and butylated hydroxyanisole (table 11).

Concentration Time of Melanocyte (M) 1 exposure 1 damage

Protecting agents Several agents were investigated for their possible protective action against

cytotoxic damage caused by 4-hydroxyanisole (figs 12 and 13). These agents were added to the culture medium in the concentrations

shown (table 111) and added to the cells for 30 min. before the addition of

TABLE I1 Comparative effects of substituted phenols

I I I

1-isopropyl- 3,4- catechol

1-tertiary butyl- 3,4-

Chydroxyanisole 2-hydroxyanisole 3-hydroxyanisole

3-tertiary butyl-4- hydroxyanisole

catechol

l x 10-3

1 x 10-6 1 x 10-4

1 x 10-3 i x 10-5

1 x 10-3

1 x 10-3

5x 10-3

1 x 10-3

5 X 10-6

30 min. I5 min. 12 hr

30 min. 30 min.

30 min. 30 min. 30 min.

30 min. 30 min.

+++ ++ + +++ + +++ - -

4-hydroxyanisole. The medium containing the 4-hydroxyanisole to be added also contained the protective agents throughout the course of the experiment.

Tyrosinase inhibitors. Copper-binding substances employed as tyrosinase inhibitors were the dipeptide glycylglycine, glutathione, cysteine and sodium diethyl-dithiocarbamate (fig. 12). The results are shown in table 111.

Free radical scavengers. A number of protecting agents are known that interrupt lipid peroxidation by trapping free radicals. They include the following substances that were tested in these experiments : glutathione, ubiquinone-6 (fig. 13) and ascorbate. The results are summarised in table 111.

Electron spin resonance studies A large electron spin resonance signal was present in control material with

a “ g ” value of 2.0020 and a line width of 18 G. This signal is ascribable to melanin (Blois, Zahlan and Maling, 1964; Pathak and Stratton, 1968). The signal obtained from treated skin was about 3 x the magnitude of the control and gave a “ g ” value of 2.0015 with a line width of 20 G. The spectra and the calculated difference are shown in fig. 14.

HYDROXYANISOLE AND PIGMENT-CELL TOXICITY 167

Concentration of

substance added (M)

TABLE I11 Effect of protective agents against damage caused by 4-hydroxyanisole

applied for 30 min.

Protection* Concentration

of Chydroxyanisole (MI

1 x 10-3

1 x 10-3

1 x 10-3

1x10-3

1 x 10-2

5 X 10-5

1 x 10-2

1 x 10-3

5 x 10-3

Addition

GI ycylgl ycine

Glutathione Glutathione

Cysteine

Ubiquinone-6 Ubiquinone-6

Sodium diethyl-dithiocarbamate Sodium diethyl-dithiocarbamate Sodium diethyl-dithiocarbamate

Ascorbate Aswrbate

__

1 x 10-3 1 Yes

1x 10-3 1 x 10-4

1 x 10-4

4x 10-3

1 x 10-4 IX 10-4 1 x 10-4

1 x 10-3 i x 1 0 - 3

I X 10-6

Yes Yes

No

No Yes

No No Yes

Yes Yes

* Protection is defined as total lack of morphological damage as seen in the light microscope when extensive melanocyte damage was observed in controls treated with 4-hydroxyanisole alone.

.,---Difference

I I 3.2 3.3 kG

3.307 g 2.0023

FIG. 14.-Electron spin resonance readings from normal untreated black guinea-pig skin and skin treated with 20 per cent. 4-hydroxyanisole for 8 days prior to biopsy showing the difference in the spectra.

168 P. A. RILEY

DISCUSSION The preferential uptake of tritiated 4-hydroxyanisole into melanocyte

cytoplasm rich in melanosomes and the similarity of the distribution to that of the dopa reaction product is evidence that 4-hydroxyanisole is acting as a substrate for tyrosinase. The product is to some extent incorporated into the pigment granules, as evidenced by the appearance of labelled granules in keratinocytes after 12-24 hours’ incubation. This may be due to the trapping of some of the radicals by reaction with the constrained radicals of melanin (Commoner, Townsend and Pake, 1954). Dose-dependent cytotoxic effects produced by 4-hydroxyanisole have been discerned by direct observation of cultured cells. The effect is selective towards melanocytes in mixed cultures containing keratinocytes and pigment cells, and unpigmented melanocytes are also spared. Similar results were found when other substrates for tyrosinase were used, but 2-hydroxyanisole and 3-hydroxyanisole, which are poor sub- strates for tyrosinase (Riley, 1969b), were not toxic. All this evidence suggests strongly that the melanocytotoxic effect is exerted by a product of tyrosinase oxidation. This is also suggested by the protection afforded by the copper- binding tyrosinase-inhibitors glutathione and sodium diethyl-dithiocarbamate. Glycylglycine was also used as a copper-binding agent and was a very effective protector against melanocyte damage by 4-hydroxyanisole in equimolar amounts, but later tests showed that glycylglycine has a negligible inhibitory effect on mushroom tyrosinase in vitro and the mechanism whereby the dipep- tide affords protection is therefore obscure. Since cysteine in 1 0 - 4 ~ con- centration failed to protect it is possible that the effect of glutathione is by a radical scavenger mechanism rather than by tyrosinase inhibition. Ascorbate was very effective in preventing melanocyte damage by high concentrations of 4-hydroxyanisoleY and this effect was considered to be due to its anti-oxidant effect. Ubiquinone-6 in low concentration was protective, but damage was increased in some cells treated with 4-hydroxyanisole and ubiquinone-6 in high concentration. This crossover effect is further evidence that radical reactions are involved. Similar conversion from anti-oxidant to pro-oxidant activity with increasing concentration of the scavenger has been reported by Matsushita, Ibuki and Aoki (1963) using a-tocopherol.

The evidence from the electron spin resonance spectra of in-vivo treated skin samples indicates that a new species of free radical is formed in epidermis treated with 4-hydroxyanisole. In the light of this evidence it seems more than probable that cutaneous depigmentation produced by phenolic substances of the class previously described (Riley, 1969a and b) is brought about by the synthesis of a diffusible free radical that can initiate lipid peroxidation in melanocytes and thus destroy them. The possible uses of antimelanocyte compounds of this kind in the treatment of malignant melanomas that retain tyrosinase activity is a subject for urgent investigation.

SUMMARY Evidence is presented that normal guinea-pig melanocytes cultured in vitro

selectively take up 4-hydroxyanisole into melanosomes. This selective effect

HYDROXYANISOLE AND PIGMENT-CELL TOXICITY 169

is a function of the state of pigmentation of the cells and their tyrosinase activity. Heavily pigmented cells show strong uptake of 4-hydroxyanisole. At 10 - 3 ~ concent rations this material is extremely toxic to melanocytes, which undergo cytoplasmic blebbing and rupture of the cell membrane in a 30-min. period. A similar effect was noted for para-substituted phenols and catechols, but 2-hydroxyanisole and 3-hydroxyanisole were without this effect. Protection against 4-hydroxyanisole toxicity was noted when free-radical scavengers (ascorbate, glutathione and ubiquinone-6) and tyrosinase inhibitors (glycylglycine and sodium diethyl-dithiocarbamate) were present in the culture medium. Electron spin resonance data on black guinea-pig skin treated in vivo with 4-hydroxyanisole indicate that a free radical is formed. It is suggested that this molecular species is a tyrosinase oxidation product of 4-hydroxyanisole that causes cell damage by initiating lipid peroxidation.

The work reported here was carried out during the tenure of a Beit Memorial Fellowship and was supported by the Medical Research Council. I thank Dr T. F. Slater for advice and for carrying out the electron spin resonance measurements, which were made possible by the assistance of Mr S. R. Tanner, Mr P. M. Butcher and Mr J. W. R. Cook of Decca Radar Ltd.

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