HYDROXYANISOLE DEPIGMENTATION: IN-VITRO STUDIES

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

University Street, London

IN the previous paper (Riley, 1969) it was concluded that the depigmenting effect of para (p)-hydroxyanisole was unlikely to be caused by a specific primary action on melanogenesis, but that it could be a secondary result of a cytotoxic action. This paper reports in-vitro studies of the toxic effects of the 3 structural isomers of hydroxyanisole to determine to what extent such effects correspond to their different depigmenting action.

MATERIALS AND METHODS

The materials used were as described in the previous paper (Riley, 1969). In addition 3,4-dihydroxyphenylalanine (G. T. Gurr), 3-methoxyadrenaline (Sigma Chemical Co.), and p-hydroxyazobenzene (British Drug Houses Ltd) were used.

14C-leucine and 3H-uridine were supplied by the Radiochemical Laboratories, Amersham. Protein synthesis. The effects on protein synthesis in rat liver slice preparations were

studied by 14C-leucine incorporation into extractable protein as the index of synthetic rate. The experimental conditions were as described by Clifford and Rees (1966). The hydroxy- anisole isomers were made up in appropriate concentrations in distilled water and neutralised with dilute sodium hydroxide. Neutralised distilled water was added to the control incub- ations. The effect of hydroxyanisole was also investigated on isolated rat liver microsomes with added ATP and P-enol pyruvate-pyruvate kinase as energy source by the method of Rendi and Hultin (1960).

RNA synthesis. Studies of RNA synthesis were carried out with cultures of HeLa cells. Tritiated uridine was added in a concentration of 0.05 ,uCi per ml to monolayer cultures grown at 37°C on medium 199 (Burroughs Wellcome) with 2 per cent. bicarbonate supplemented with 10 per cent. calf serum (Oxoid) and equilibrated on serum-free medium for 3 hr. The test substances were added 1 hr beforehand. The cells were harvested after incubation for 1 hr. The RNA was extracted by the method of Scherrer and Darnel1 (1962) and the specific activity of the total RNA extracted determined. Optical density was meas- ured at 260 nm and radioactivity measured in a Packard Scintillation Spectrometer with a BBOT (Ciba) scintillator in a thixotropic mixture containing the samples.

Mitochondria1 respiration. The effects on mitochondria1 respiration were studied by the neotetrazolium reduction procedure of Slater (1963). Rat liver mitochondria were isolated from 1 g wet wt of liver into 10 m l 0 . 2 5 ~ sucrose by the method of Slater (1966) and 0.1 ml of this suspension was added to succinate-EDTA-phosphate buffer. After 2 minutes’ incub- ation at 37°C 0.15 ml of 1 per cent. neotetrazolium chloride was added, and the reaction was stopped after 10 min. with 2 ml 10 per cent. TCA. The tetrazolium was extracted in 4 ml ethyl acetate and the extinctions read at 510 nm. Antimycin A (Sigma) was made up in

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

J. PATH.-VOL. 97 (1969) 193 0

194 P. A . RILEY

ethanol for addition to the tubes and the ethanol removed by gentle warming. Hydroxy- anisoles were made up in water and neutralised with NaOH before addition.

Tyrosinuse activity. The majority of experiments were made with mushroom tyrosinase (Sigma Grade 11) in a concentration of 0.1 per cent. in 0 . 1 ~ phosphate buffer (PH 7.4). A crude tyrosinase-containing extract of guinea-pig epidermis obtained by washing the separated epidermis in a small volume (1-2 ml) of Triton XlOO (0.1 per cent. in water), which extracts

I I I 1 I l l 1 I I I I 1 I I I I I i I I I l l I I i I I I I

2.c

1.5 v > U U n U

U W

lA

._

._

E

a 1.0

0.5

0.1 1.0 Concentration (mM)

FIG. 1.-Effect of hydroxyanisole on protein synthesis. The plot shows the effect of increasing concentrations of hydroxyanisole on the ratio of the specific activity of extracted protein com- pared with control values. The points are in each case the mean of 2 experiments. Mean specific activity of controls = 595 c.p.m. per mg dry wt of protein (= 1.0). 0 = o-Hydroxyanisole; 0 = in-hydroxyanisole; V = p-hydroxyanisole.

tyrosinase from the basal melanocytes (Riley, 1966), was also employed. Activity was measured by rates of oxygen uptake with an oxygen electrode, and by rates of product formation measured by increases in ultraviolet absorption at the product absorption maxima with a Unicam SP800 spectrophotometer. In experiments with the epidermal extract the incubation temperature was 37°C. All other experiments were carried out at room tem- perature (c. 24°C).

RILEY PLATE LXXX

HYDROXYANISOLE DLPIGMENTATION

FIG. 5a.-Tncubated in dopa medium.

FIG. 5b.-ATP reaction product.

FIG. 5.-Adjacent sheets of separated epidermis, removed 2 days after cessation of 3 weeks’ treatment with p-hydroxyanisole. x 420.

RILEY PLATE LXXXI

HYDROXYANISOLE DEPIGMENTATION

FIG. 6a.-From animals treated with o-hydroxyanisole.

FIG. 6b.-Frorn animals treated with rn-hydroxyanisole.

FIG. 6c.-From animals treated with p-hydroxyanisole.

FIG. 6.-Vertical sections of skin from the hairless region behind the ear, removed 1 wk after cessation of 3 weeks' treatment with a hydroxyanisole. Left, incubated in dihydroxyphenylalanine medium; right, incubated in ATP medium. x 235.

HYDRO X YA NISOLE DEPIGMENTA TI0 N 195

Identification of adrenaline oxidation product was made by converting the product to the corresponding semicarbazone by the method described by Vogel (1948) and comparing the spectrum with that of adrenochrome semicarbazone obtained from Sigma Chemical Co.

Haernolysis experiments. The effect of hydroxyanisole on erythrocyte membranes was tested on packed human red cells resuspended in 10 vol. of 0.9 per cent. saline. Ortho-, meta- or para-hydroxyanisole made up in saline was added to give final concentrations of between 1 and 20 m ~ , and 0.5 ml tyrosinase (0.1 per cent. in 0.9 per cent. saline) was added to the tubes in parallel experiments to a final vol. of 2 3 ml. Controls were made up to equivalent volumes with saline. Incubation was for 1 hr at 37°C and the reaction was stopped by bubbling coal gas through. The intact cells were sedimented by centrifugation (9009 for 1 min.) and the extinction of the supernatant was read at 538 nm. Corrections were made for the absorption of hydroxyanisole oxidation product obtained from incubation mixtures not containing the red cell suspension.

RESULTS Inhibition of protein synthesis

The results in fig. 1 show that at concentrations greater than l m ~ all 3 forms .of hydroxyanisole have an extremely powerful inhibitory effect on protein synthesis. In liver slice preparations a concentration of 5 m ~ led to about 80 per cent. inhibition of 14C-leucine incorporation. At higher concentrations a saturation effect was produced and at values below l m ~ stimulation was ob- served. The ortho- form was found to be less inhibitory than meta- and para- hydroxyanisole both in liver slice preparations and in the isolated microsomes.

Eflect on ribonucleic acid synthesis The results shown in table I indicate that under the conditions of the experi-

ment about a 50 per cent. inhibition of RNA synthesis is produced by hydroxy-

TABLE 1

Specific activity of RNA extracfs from HeLa cell cultures treated with hydroxyanisole

Specific activity, as percentage of controls, Final molarity 1 of RNA extracts treated with

of hydroxyanisole (1nM)

1 o-hydroxyanisole m-hydroxyanisole p-hydroxyanisole I

107 50 56

I 1 27 84 I 5

Specific activity of controls: 2110 and 1300 (c.p.m. x 5/OD) respectively.

anisole in a concentration of 5 m ~ . Ortho-hydroxyanisole was found to be more inhibitory than the meta and para forms and produced a 16 per cent. inhibition of total RNA synthesis in a concentration of 2 m ~ , at which level m- and p-hydroxyanisole failed to produce any inhibition in comparison with controls in which equivalent volumes of distilled water were added to the culture medium.

196 P. A . RILEY

TABLE 1I Effects of hydroxyanisole on neotetrazolium reduction by

isolated mitochondria

System Formazan produced*

Control Antimycin o-Hydroxyanisole o-Hydroxyanisole+ Antimycin m-Hydroxyanisole m-Hydroxyanisole+ Antimycin p-Hydroxyanisole p-Hydroxyanisole+ Antimycin

43 16 33 0

16 1

14 3

* Calculated from the mean extinctions at 510 nm of ethyl acetate extracts of the reaction system The final molarities of the additives were 0.5 mM for after 10 minutes’ incubation at 37°C.

hydroxyanisole and 0.001 mM for Antimycin A.

I I I I - p-Hydroxyanisole - Oxidised-Fenton’s reagent

0 .

- 0 .

250 300 Wavelength (nm)

FIG. 2.-Ultraviolet absorption spectra. m = p-Hydroxyanisole. p1 = Primary product of p- hydroxyanisole oxidation by mushroom tyrosinase. pz = Secondary oxidation product of p- hydroxyanisole. p3 = Absorption spectrum of final tyrosinase oxidation product of p-hydroxy- anisole. f = Absorption spectrum of product of p-hydroxyanisole oxidised with Fenton’s reagent.

HYDROXYANISOLE DEPIGMENTATION

TABLE I11 Comparative maximum rates of oxidation by mushroom tyrosinase

Substrate

Dihydroxy pheny lalanine Dihydroxymandelic acid Noradrenaline Adrenaline o-Hydroxyanisole 3-Mcthoxymandelic acid 3-Met hoxynoradrcnaline 3-Mcthoxyadrenaline m-Hydroxyanisole p-Hydroxy phcnylpyruvate p-H ydroxyanisole I--Tyrosinc

p-Hydroxyacetophenone

p-Hydroxybenzoic acid

Rate 0 2 uptake

34.5 10.0 22.5 7.5 1.1

<0*01 <0.01 t0.01

1.8 13.0 9.0 6.0

0.9

0.01

-

OD primary product

absorption maximum

1.4 0.28 0.24 0.53 ... ... ...

0.05 0.13 093 0.12

0.10

...

Maximum absorption

band of primary product

(nm)

305 343 290 305 ... ... ... ...

245 340 265 280

273

...

Maximum absorption

band of secondary products (W

general 253 253 260 ... ... ... ... ...

240 286

general 305

general 260 ...

197

Oxygen consumption was measured by oxygen electrode and the rates expressed in per cent. uptake per min. at 24°C. The reaction mixture consisted of 1 ml 0 . 0 1 ~ substrate and 0.1 ml 0.1 per cent. tyrosinase in 0 . 1 ~ phosphate buffer at pH 7.4. Rates of product formation are given in terms of the increase in absorbance per min. at the absorption maximum for the primary product. Substrate concentration 0 . 2 m ~ , enzyme concentration 0.01 per cent. All estimations were made on reaction mixtures in 0 . 0 1 ~ phosphate buffer (PH 7.1) at 24°C.

TABLE IV Effect of p-hydroxyanisole on oxidation of tyrosine and dopa

by mushroom tyrosinase

Concentration (mM) Rate found Rate calculated

y u L " n J - .:",.l'> , 1 at 320nm at265nm

3.3 0.3 5 0.3 5 0.3 3.3 ... ..I

...

...

0.3 0.3 5 3.3 ... ... ... 3.3 0.3 5 0.3

...

...

...

... 5 0.3 0.3 0.3 0-3 5 3.3

0.4 1 . 1 1.6 0.4

22.5 1.8 1.5 1.5 1 .o I -2 0.7

...

...

...

... 1.2

35.5 0.9 0.9 2-1 0.9 1.2

~

Maximum rates estimated as the increase in optical density per min. The calculated rates were obtained by addition of the rates found when the substrates were incubated separately with tyrosinase. All estimations were made in phosphate buffer a t pH 7.4 at 24°C. Enzyme concentration = 0.005 per cent.

198 P. A. RILEY

Effects on respiration by isolated mitochondria The results in table I1 indicate that in each instance considerable inhibition

of neotetrazolium reduction occurred prior to the point of interaction of Anti-

A

c 0

n ._ bJ

E 2 u <J

2 V 0 L n L 0

€3

2 Do?a

Tyrosin-

p-Hydroxyanisole 0

i

Substrate concentration fmM\

FIG. 3.-(A) Effect of substrate concentration on the initial rates of formation of tyrosinase oxidation products of tyrosine (rate of increase in absorbance at dihydroxyphenylalanine absorption maximum); and dopa (rate of increase in absorbance at 320 nm).

@) Effect of substrate concentration on the initial rates of formation of tyrosinase oxidation products of tyrosine (measured at 320 nm) andp-hydroxanisole (measured at 265 nm). Tyrosine rates x 10. Tyrosinase concentration = 0,005 per cent.

mycin A, and suggest, therefore, that hydroxyanisole blocks the respiratory chain prior to cytochrome c (Slater, 1963), possibly by interference with the ubiquinone-requiring step (Hoffmann-Ostenhof, 1963).

HYDROXYANISOLE DEPIGMENTATION 199

Oxidation of hydroxyanisole by tyrosinase It was found that mushroom tyrosinase rapidly oxidises p-hydroxyanisole

with the production of a primary product with an absorption peak at 263 nm and a final melanin-like product with linear absorption in the ultraviolet range (fig. 2). Under similar conditions m-hydroxyanisole was slowly oxidised giving a yield of primary product with an absorption peak at about 245 nm; o- hydroxyanisole was not oxidised at all under these conditions. These rates are compared with the rates of oxygen uptake in table 111.

Efect of p-hydroxyanisole on tyrosine-tyrosinase and dopa-tyrosinase systems The results of kinetic studies conducted with mushroom tyrosinase are

shown in table ZV and figs. 3 and 4. These indicate that oxidation of both

c 0 U m .-

E '2

2

U 0 3 -0

a c 0 a, U

2

p-Hydroxyanisole 0 Tyrosine

FIG. 4.-Effect of tyrosinase concentration on the initial rate of product formation from tyrosine and p-hydroxyanisole. Tyrosine product measured at 320 nm (x 5). Hydroxyanisole product measured at 265 nm. Substrate concentration = 0.8 m ~ .

p-hydroxyanisole and tyrosine is increased when both substances are present together in the medium, but that whereas p-hydroxyanisole is more rapidly oxidised in the presence of dopa, dopa is more slowly oxidised under these conditions. The addition of equimolar amounts of m-hydroxyanisole caused a 50 per cent. reduction in the rate of tyrosine and dopa oxidation. Ortho- hydroxyanisole had no effect on either tyrosine or dihydroxyphenylalanine oxidation, but led to the retention of the 265 nm peak and concurrent reduction

200 P. A . RILEY

Rate difference

(1st min.) Substrate plus extract

in the 305 nm absorption during the oxidation of p-hydroxyanisole, presumably by a non-enzymatic reaction with the second oxidation product of p-hydroxy- anisole. On prolonged incubation a new absorption maximum at 276 nm appeared.

Rate difference Ratio

plus substrate*

Hours FIG. 5.-Oxidation of tyrosinase substrates by an extract of guinea-pig epidermis. Absorbance

measured at 305 nm for dopa, 280 nm for tyrosine and at 265 nm for p-hydroxyanisole.

TABLE V Rate of oxidation by epidermal extract

Dopa Tyrosine o-Hydroxyanisole m-Hydroxyanisole p-Hydroxyanisole

0.74 0.0 I ... -0.07 0.23 1 .O

0.0 0.02 0.09 0.0 0.02 0.09

-0.078 0.35 1.52

Maximum rates determined as increase in absorption at 305 nm per min. ( x 10) at 40°C. Substrate concentration 8 m ~ . The tyrosinase-containing extract was added in a vol. of 0.4 ml to 3 ml of reaction mixture. The figures show the mean values of 3 experiments using different extracts.

* Maximum rate of dopa-extract system containing substrate minus the rate for dopa-extract system alone.

Tyrosinase-containing extract from guinea-pig epidermis Comparisons of ultraviolet absorption spectra before and after overnight

incubation at 40°C showed that both tyrosine and p-hydroxyanisole were

HYDROXYANISOLE DEPIGMENTATION

Treatment of epidermal extracts

TABLE VI Comparison of absorption spectra of hydroxyanisole-treated

and untreated epidermal extracts

Absorption at

~- I

None o-Hydroxyanisole m-H ydroxy anisole p-H ydrox yanisole

I 245 nm I 265nm

0.13 0.25 0.14 0.27 0.20 0.40 0.11 0.25

201

Concentration of o-hydroxyanisole (mM) FIG. 6.-Effect of hydroxyanisole with and without tyrosinase on lysis of erythrocytes.

(A) o-Hydroxyanisole. I . PATH.-VOL. 97 (1969) P

202 P. A. NLEY

oxidised by the epidermal extract. The rate of oxidation was, however, too slow (fig. 5 ) to enable the initial velocities to be estimated accurately. The technique employed therefore was to compare the rate of change in spectral absorption of solutions of tyrosine and hydroxyanisole in substrate-extract systems containing dihydroxyphenylalanine. The results (table V) show that

U r

aJ U

L U R

Concentration of m-hydroxyanisole (mM)

FIG. 6.-Effect of hydroxyanisole with and without tyrosinase on lysis of erythrocytes. (B) m-Hydroxyanisole.

both tyrosine and p-hydroxyanisole autoxidation (which proceeds at rates of 0.06 and 0.07 respectively under the experimental conditions used) is initially inhibited by the extract. In the presence of dopa, however, these substances are rapidly oxidised; p-hydroxyanisole is oxidised at about one-and-a-half times the rate for tyrosine determined at 305 nm. In order to check whether hydroxy- anisole oxidation occurs in viva, extracts of 20 mg (wet wt) of separated

HYDROXYANISOLE DEPIGMENTATION 203

epidermis from treated animals were made in 3 ml 0.9 per cent. saline and the ultraviolet spectra compared with similar extracts of untreated skin. The results (table VI) indicate that only the oxidation products of m-hydroxyanisole are detectable to a significant extent.

100

10

L)

c a, U

1

Concentration of p-hydroxyanisole (mM) FIG. 6.-Effect of hydroxyanisole with and without tyrosinase on lysis of erythrocytes.

(C) p-Hydroxyanisole.

Other ring-substituted phenols as substrates for mushroom tyrosinase A range of ring-substituted phenols was tested as potential substrates for

mushroom tyrosinase. The rates of oxidation are shown in table 111. The oxidation product of adrenaline was identified as adrenochrome.

Eflect on the erythrocyte membrane system The results are summarised in fig. 6, which shows that o-hydroxyanisole

has a greater haemolytic action than m- and p-hydroxyanisole; this effect is not

204 P. A . RILEY

71 84 85

influenced by the addition of tyrosinase to the system whereas (except at higher concentrations in the case of m-hydroxyanisole) the presence of enzyme increases the haemolysis due to m-hydroxyanisole and p-hydroxyanisole. The control values were in the region of 0-5 per cent. of the total osmotically produced haemolysis.

DIscussroN The results of the experiments on ribonucleic acid and protein synthesis

and mitochondrial respiration would seem to indicate that although the isomeric

TABLE VII

Comparison of depigmenting action with in-vitro efsects of 5 mM ortho-, meta- and para-hydroxyanisole

24 36 31

I ! Percentage inhibition of

Inhibition of mitochondrial

respiration (per cent.)*

Substance

Depigmenting action Substance

synthesis

Haemolysis I Rate of I Oxidation of tyrosinase oxidation by 1 by epidermal

(per cent.) tyrosinasef extract Haemolysis in presence (per cent.)

I

o-Hydroxyanisole rn-Hydroxyanisole p-Hydroxyanisole

- + ++++

27 50 56

protein protein synthesis synthesis

by liver slice ~ by microsomes

o-Hydroxyanisole ~ 24 1 2.6 2.6 1 1.1 1 0.09 rn-Hydroxyanisole 1 1 .5 ~ 2.7 1.8 I 0.09 p-H ydroxyanisole

~ 6": I 0.9 4.4 I 9.0 I 152

* 0 3 ' m ~ . i- 10mM. Expressed as rate of oxygen uptake.

forms of hydroxyanisole are all toxic to these systems in vitro there is no obvious direct relation between these effects and their relative depigmenting action (table VII).

On the other hand, the in-vitro experiments with tyrosinase show that a relation may exist between depigmenting activity and the extent to which the hydroxyanisoles act as substrates for the enzyme. Reasons have already been given why this is unlikely to come about by competitive inhibition (Riley, 1969), and there is additional evidence from this study that this is not the case, since p-hydroxyphenylpyruvate and adrenaline and noradrenaline, which are rapidly oxidised by tyrosinase in vitro and therefore would be expected to act as competitive inhibitors of the enzyme, failed to produce in-vivo depigmenta- tion and also, the data shown in table IV indicate that in vitro the effect of

HYDRO X YANISOLE DEPIGMENTA TION 205

p-hydroxyanisole is to stimulate tyrosine oxidation. The nature of this effect, which is cooperative, is not clear.

The data regarding the formation of products from p-hydroxyanisole by tyrosinase oxidation indicate that the final product @3) is a melanoid polymer and the sequence of reactions probably parallels the oxidation of tyrosine:

OH

0 0

CH3

(PI) OH

It is possible that by reduction of the quinone oxidation product (p2) a semiquinone free radical (p4) is formed. If the rate of polymerisation were slow these radicals might diffuse out of the melanosomes and take part in chain initiation of lipid peroxidation resulting in damage to cellular organelles (Slater and Riley, 1966) and selective destruction of melanocytes. The results of the haemolysis experiments would support this suggestion.

SUMMARY The results of an investigation of the properties of ortho-, meta- and para-

hydroxyanisole in vitro are reported. All these substances were found to inter- fere with ribonucleic acid and protein synthesis and with mitochondria1 respira- tion to a similar degree, in contrast to their depigmenting action in vivo. A correlation was found between this action and the ability of the hydroxyanisole isomers to act as substrates for tyrosinase, and to cause red blood cell lysis in the presence of this enzyme.

It is a great pleasure to thank Professor C. Rimington for his encouragement and guidance throughout this study. I thank Dr K. R. Rees, Dr T. F. Slater and Professor J. D. Judah for their help and many valuable discussions. I am indebted to Mr V. K. Asta for drawing the figures.

REFERENCES CLIFFORD, JANET I., AND REES, K. R. HOFFMANN-OSTENHOF, 0. . . . 1963. 112 Metabolic inhibitors, ed. by R. M. Hoch-

ster and J. H. Quastel, New York, vol. 2 , p. 145.

1966. Biochem. J. , 102, 65.

RENDI, R., AND HULTIN, T. . . . 1960. Expl cell Res., 19,253.

206 P . A . RILEY

RILEY. P . A . . . . . . . . 1966 . Br . J . Derm., 78. 559 . 1962 . Biochem . Biophys . Res . Commun., 7.486 .

SLATER. T . F . . . . . . . . 1963 . Biochim . biophys . Acta. 77. 365 . 1966 . Excerpta med., Znt . Congr . Ser., 115. 30 .

VOGEL. A . I . . . . . . . . 1948 . Practical organic chemistry. London. p . 342 .

.. . . . . . . . 1969 . J . Path., 97. 185 . SCHERRER. K., AND DARNELL. J . E . .

.. . . . . . . . SLATER. T . F., AND RILEY. P . A . . 1966 . Nature. Lond., 209. 151 .