The Journal of Pathology

Vol. 114 No. 1

T H E ROLE OF SUBSTITUTED ANISOLES I N EPIDERMAL MICRO-INVASION

P. A. RILEY AND P. SEAL* Department of Biochemical Pathology, University College Hospital Medical School and

Imperial Cancer Research Fund Laboratories, London

PLATES 1 AND 11

ANT I o x I D A N T s of the phenolic series are widely used in the preservation of food. The addition of these substances to foodstuffs is reviewed from time to time by a government-appointed committee (HMSO, 1963). The permitted antioxidants have been tested for carcinogenicity in animal feeding experiments and no evidence of increased incidence of tumours has been reported (Wilder and Kraybill, 1948; Brown et al., 1959; Hodge et al., 1964; Clegg, 1965; Gilbert and Golberg, 1965). Butylated hydroxytoluene (BHT), in common with some other antioxidants, has been shown to increase the life expectancy of animals given long-term supplements in their diets (Harman, 1968a and b; Comfort et al., 1971). Although it is not certain to what this effect can be ascribed the evidence against any tumour-promoting action by, at least a range of anti- oxidants, appears strong.

Paradoxically, when a series of antioxidant derivatives of hydroxyanisole were tested by topical application to the skin and cheek-pouch epithelium of animals they were shown to produce unusual morphological lesions in the cells of the generative basal layer of epithelium (Riley and Seal, 1968 ; Riley and Seal, 1969; Seal et al., 1969; Woods and Smith, 1969a and b). The principal lesion was the production of pseudopodial extensions of the basal cell cytoplasm which extended through the normally limiting basal lamina of the epithelium into the connective tissue below. This phenomenon is termed micro-invasion and has been observed in early epithelial tumours of natural origin (Hinglais-Gillard et al., 1961 ; Frei, 1962; Sugar and Farago, 1966; Olson et al., 1968; SugBr, 1968; Frithiof, 1969; Cohen et al., 1971), and in the early stages of tumours experi- mentally induced by the local application of DMBA (7,12 dimethylbenz-(a)- anthracene) to the cheek-pouch epithelium of hamsters (Woods and Smith,

Received 18 July 1973; accepted 19 Oct. 1973. * Present address: Elin Laboratories, Shap, Penrith, Cumbria.

J. PATU. VOL. 114 (1974) A

2 P. A . RILEY AND P. SEAL

1969a; Woods and Smith, 1970). The significance of micro-invasion is not clear since it has been observed in psoriatic skin (Cox, 1969) and in healing wounds (Odland and Ross, 1968; Ross and Odland, 1968). On the other hand, micro- invasion is not a concomitant of epithelial hyperplasia produced by the appli- cation of irritants such as benzene and turpentine (Tarin, 1968). It seems, therefore, that derivatives of 4-hydroxyanisole (4-OHA) do not act as simple irritants to the epithelium but mimic one action of a known carcinogen (DMBA). Because this raises important questions about the biological action of phenolic antioxidants, the effects of a range of substituted phenols have been investigated together with some non-phenolic antioxidants in an attempt to gain insight into the mechanism of action of the hydroxyanisoles.

MATERIALS AND METHODS

Application The agents which are tabulated (tables I1 and 111) were made up in lanolin. With the

exception of Promethazine (4 per cent.) and u-tocopheryl-ethylene glycol 1000 succinate (5 per cent.) all the substances were made up as 20 per cent. creams. These were kept at 4°C and applied daily 5 times a week to the backs of the ears of male guinea pigs of an inbred black strain. All animals were fed with a standard pellet diet (MRC Diet SGI) and water ad libitum.

Biopsy and Processing Thiersch biopsy samples were taken freehand from 53 animals under halothane anaes-

thesia. The animals were kept in pairs in separate cages according to the application to be tested. The agent was applied to both ears and, in general, biopsies were made in rotation so that 4 wk elapsed between successive biopsies of the same ear. although in some instances a shorter period intervened. In all cases care was taken to leave a margin of at least 1 cm between the zone of biopsy and the previously sampled site. The sample material consisted of epidermis and the superficial dermis in sheets approximately 4 m m 2 in surface area. The material was fixed in osmium tetroxide in 0 . 1 ~ phosphate buffer CpH7.4) at 0°C for 60 min., washed in buffer, dehydrated and embedded in Araldite. Sections were cut with an LKB Ultramicrotome, stained with uranyl acetate and lead citrate and examined in a Siemens Elmiskop 1 electron microscope.

Time-course The time-course of the experiments was in most cases over a treatment period of 6 wk.

Biopsies were performed at weekly intervals during the treatment period (in some cases more frequently at the commencement of treatment) and at Cdaily intervals for 2 wk after the cessation of treatment.

Sample size and Discrimination In each case the number of sections examined in the electron microscope was sufficient to

give an observed length of basal lamina of more than 3 mm. The section thickness was about 500 A. Therefore, the discrimination afforded by the procedure in deciding whether or not micro-invasion was present in the sectioned material was sufficient to detect lesions in excess of one per 7.5 cell equivalents.

RESULTS Control material from animals, whether untreated or treated with the cream

base alone, showed normal epidermis with an intact basement membrane. No evidence of basal cell pseudopodial extensions was found.

INDUCED MICRO-INVASION 3

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o+++oooo 0 00000 0 0 0

* i +o++oooo 0 00000 0 0 0 + m

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++++oooo 0 00000 000 + %..

4 P. A. RILEY AND P. SEAL

By contrast it was found that a number of the substituted phenols tested caused basal cell pseudopods which extended into the dermis similar to those observed in skin treated with 4-hydroxyanisole (fig. 1, table I). The substances which were found to cause micro-invasion are shown in table 11. An interesting

TABLE I1

Substances inducing micro-invasion

4-hy droxyanisole 3-hydroxyanisole* 4-tert butyl phenol 4-tert butyl catechol 4-is0 propyl catechol 7C-phenyl 4-hydroxyanisole propyl gallate 2-tert butyl4-hydroxyanisole 3-tert butyl 4-hydroxyanisole (2, 3) butylated 4-hydroxytoluene 3-amino anisole

* after prolonged treatment.

correlation was found between the ability of the agents to depigment the skin and their induction of micro-invasion. This led to the consideration that the effect which was observed might be due either to a metabolite resulting from the enzymic oxidation of the phenols by tyrosinase (Riley, 1969a and b) or a

TABLE I11

Substances not found to induce micro-invasion

2-hydroxyanisole gallic acid 4-flUOrO anisole 3-flUOx-O anisole hydroquinone L-3,4-dihydroxyphenylalanine sodium sulphite ascorbic acid a-tocopheryl ethylene glycol-loo0 succinate promethazine hydrochloride

secondary result of melanocyte destruction (Riley, 1970) which might cause a local loss of tissue homeostasis (Riley, 1971). However, when the unpigmented skin of recessively white-spotted animals was examined after treatment with 4-hydroxyanisole the extent and apparent frequency of basal cell micro-invasion was the same as that found in pigmented skin. Since the unpigmented skin contains neither melanocytes nor tyrosinase it must be concluded that the effect of the substituted phenols is exerted directly either on the epidermal cells or some component in the upper part of the dermis. In v i m experiments on cultured keratinocytes designed to distinguish between these modes of action, although suggestive of a direct effect on the epidermal cells, were inconclusive.

RILEY AND SEAL

INDUCED MICRO-INVASION

PLATE I

FIG. 1 .-Electron micrograph showing micro-invasion. The junctional layer is illustrated showing a pseudopod (P) projecting from the epidermis (E) through a defect in the basal lamina (arrowed) into the underlying dermis (D). The scale bar represents 1 pm.

RILEY AND SEAL

INDUCED MICRO-INVASION

PLATE I1

FIG. 2.-Electron micrograph showing myelin figures (M) in a keratocyte from skin treated with promethazine. Pigment granules (G) are present and it is possible that the “myelin figures” could be abnormal premelanosomes. The nucleus (N) and a desmosomal junction (J) are indicated. The scale bar represents 1 pm.

INDUCED MICRO-INVASION 5

A number of non-phenolic antioxidants were investigated for their ability to induce basal cell micro-invasion. In no case was evidence of such a process found (see table 111) although “ myelin figures ” were observed in the epidermal cells of the skin treated with promethazine (fig. 2).

In all instances in which micro-invasion was observed during the treatment period, basal cell pseudopods were no longer found 4 days after the treatment was stopped. Some of the treated animals have been observed for more than 2 yr after the cessation of treatment and none have developed tumours in the treated areas.

DISCUSSION The experiments indicate that when treatment with the micro-invasion-

inducing agents is stopped the skin rapidly returns to normal even after 6 weeks’ prior treatment. This test period may be too short for a neoplastic process to be entirely ruled out. However, the reversibility of the micro-invasion produced by hydroxyanisole and related compounds suggests that these substances pro- duce changes in cellular behaviour which only mimic those found in early cancer.

The effect can be excluded as being a phenomenon produced by antioxidants in general because neither ascorbate, sodium sulphite, promethazine nor vitamin E caused micro-invasion under similar test conditions. On the other hand, it seems to be a property of phenolic compounds with a non-polar side chain and a para hydroxyl group. It is possible that these compounds react with some component of the cell membrane, and may thus alter the surface properties of the affected cells. An interesting exception is 3-amino anisole, but this substance may undergo a metabolic hydroxylation to the corresponding 4-hydroxy compound.

The data would seem to exclude interactions involving hydrogen bonds either with the hydroxyl group acting as a donor or the corresponding quinone acting as an acceptor since under these circumstances one would expect either 4-aminoanisole or 4-flUOrO anisole to cause micro-invasion. It is possible that 4-hydroxyanisole and its derivatives form covalent bonds with some component of the cell membrane perhaps through a free radical reaction. Whether this is the case, and if so how it happens, remains to be elucidated.

Whilst these results might call into question the safety of the phenolic antioxidants it is important to note that the concentrations used in the topical experiments (10-20 per cent.) are much greater than those normally encountered in foods (less than 1 per cent.), the material used was in constant contact with the affected tissue and, because of the paucity of the detoxifying enzyme systems in epidermis, will have been removed only slowly from the site of action in contrast to the situation for ingested material. The local concentration of the substance is known to be critical for the induction of abnormal pseudopods since no micro-invasion was observed in skin treated with a cream containing 1 per cent. 4-OHA. Moreover, as has been pointed out (Grasso and Rostron, 1970), morphological similarity of lesions is not necessarily a reliable indication that the same pathological process is involved in their production.

6 P . A. RILEY AND P . SEAL

SUMMARY The morphological effects of a range of substituted phenols and antioxidants

applied to guinea pig ear skin have been examined with particular reference to the production of micro-invasion of the dermis by basal keratocytes. The induction of micro-invasion was found to be a property of phenolic compounds with non-polar side chains and a para hydroxyl group.

We thank Dr M. G. P. Stoker for helpful criticism and the Medical Research Council for support. The tenure of a Research Fellowship from the Wellcome Trust by one of us (P.A.R.) during the early stages of the work is acknowledged.

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INDUCED MICRO-INVASION 7

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