Albrecht v. Graefes Arch. ldin. exp. Ophthal. G r a e f e s A r c h i v 201, 207--212 (1977) ftirklinischeundexperimentelle

Ophthalmologie �9 by Springer-Verlag 1977

Phenol Oxidase Activity of the Iris and the Ciliary Body in Rats

Hern~n Valenzuela*, Christa Maria Schedtler, Hans Reinhard Koch and Otto Hockwin

Div. Biochemistry of the Eye, Clinical Institute for Experimental Ophthalmology, University of Bonn (Director: Prof. Dr. Erich Weigelin), Abbdstr. 2, D-5300 Bonn-Venusberg, Federal Republic of Germany

Summary. Phenol oxidase activity of the iris and the ciliary body in normal young rats of three differently pigmented strains, Lewis (albino), E3 (light brown, hooded),

and BDE (black, hooded), was studied using DL-Dopa. Applicabil i ty of the histo- chemical method was controlled with skin samples taken from the necks of the animals. In the pigmented strains, the appearance of newly formed pigment at the pigmented epithelium and stroma of the iris and ciliary body was observed. In the E3 animals, the reaction was slightly positive, while in the BDE rats it was more pronounced. No phenol oxidase activity was detected in the ocular structures of the albinotic rats.

Zusammenfassung. Die Phenoloxidase-Aktivit~it wurde in Iris und Ciliark6rper yon normalen, jungen Ratten bei drei verschieden pigmentierten St~immen untersucht:

Lewis (Albino)~ E3 (leicht br~iunliche Haube) und BDE (schwarze Haube). Substrat fiir die histochemische Reaktion war DL-Dopa, Kontrollen der Reaktion wurden

mit Hautschnitten vom Nacken der Tiere durchgefiihrt. Bei den pigmentierten

St~immen wurde histochemisch die Pigmentbildung im Pigmentepithel und Stroma der Iris und des Ciliark6rpers beobachtet , wobei die Farbintensit~it bei den BDE Tieren st~irker ausgepr~igt als bei den E3 Tieren war. Die Gewebe der Albinoratte zeigten keine Phenoloxidase-Aktivit~it.

I ntroduction

The mechanism of melanin formation has caught the at tent ion of many investigators. However, most of them have studied this problem in the skin (Meirowsky, 1940). Reports on ocular tissues are relatively scarce and even scarcer on the iris and ciliary body.

* Fellow of the Alexander yon Humboldt-Stiftung

208 H. Valenzuela et al.

Lillie (1956) has summarized the reported results with various substrates in the so-called tyrosinase-Dopa reaction. While using D-Dopa no positive reactions are re- ported, although there are some using DL-Dopa and sections of adult mammalian skin; however, no work on melanin formation in the eye is mentioned. Most of the authors have used L-Dopa as substrate, but mainly in the skin. Only Miescher (1923), using the Dopa reaction described by Bloch (1917), found a positive reaction in the pig- mented epithelium and in the pigmented cells of the choroid, ciliary body, and iris of the rabbit, guinea pig, and chicken during the embryonal stage. No further histo- chemical reports concerning Dopa reaction in the uvea were available.

Koch and Doldi (1975), reported that naphthalene cataract is more pronounced and develops earlier in pigmented animals, suggesting, therefore, a possible role of the anterior urea in the development of these experimental cataracts.

The aim of the present study is to make a first approach to hypothesis, checking out the phenol oxidase activity of the iris and ciliary body using DL-Dopa as sub- strate in young rats of the same strains used by Koch and Doldi.

Materials and Methods

Normal young rats (Central Institute for Test Animal Breeding, Hannover, FRG) were used in this study. They belonged to the following strains: Lewis (albino), E3 (light brown, hooded) and BDE (black, hooded).

Fifteen animals were used, five of each strain. Thirteen of them were between 14- and 18/days-old and their weight varied from 17.6 to 27.5 g. One E3 rat was 31-days- old (58.1 g.) and one Lewis rat was 41-days-old (118.5 g.).

In five experiments, three rats (one of each strain) were killed with ether. The right eye was enucleated and skin samples were taken from the animals necks. The tissues were frozen quickly with solid carbon dioxide and serial cryostat sections with a thickness of 10 microns were fixed for five min in 4% buffered formalin at 4 ~ C.

Sections of the three different skin samples and eyes were incubated simultane- ously at 37 ~ C for 90 min in the following solutions, freshly prepared before each experiment: (1) 50 ml 0.1 M phosphate buffer, pH 7.4; (2) 50 ml 0.1 M phosphate buffer, pH 7.4 plus 50 mg DL-Dopa (5.07 mM 3-4 dihydroxyphenylalanine); (3) 50 ml 0.1 M phosphate buffer, pH 7.4 plus 50 mg DL-Dopa and 11.25 mg (1 mM)

sodium diethyldithiocarbamate. Different times of fixation with 4% formalin had no influence, but the reaction

was negative if the fixation was not adequately cooled.

Results

Clear differences were found in the skin of the three strains. In the presence of DL- Dopa, the black skin of the BDE animals showed a remarkable positive reaction with many pigmented cells situated in the dermis, while the light brown skin of the E3 rats had a less positive reaction, and the albino skin of the Lewis strain presented only a

Phenol Oxidase Activity 209

30 rn t I

Fig. 1. Iris section of a Lewis albino rat, incubated in 5mM DL-Dopa at pH 7.4 for 90 min, showing no traces of induced pigment. No counterstain

slight positive one. No pigmented cells were seen in tissues incubated in solutions (1) and (3) in any of the three strains.

With respect to the iris and ciliary body, in the Lewis rats no phenol oxidase activity was detected (Fig. 1).

While in the E3 animals the reaction was slightly positive, in the BDE rats the appearance of newly formed pigment at the pigmented epithelium and stroma of these ocular structures was clearly observed (Figs. 2-3). The difference was particu- larly evident when comparing the preparations incubated in solutions (2) and (3), that is, with or without sodium diethyldithiocarbamate. However, comparing the prepara- tions incubated in solutions (1) and (2), the difference, although present, was smaller.

Discussion

The problem of enzymatic participation in melanin formation has actually not yet been thoroughly elucidated. It is known that besides phenoloxidase, Dopa can also be oxidized by peroxidase or by the cytochrome system. The action of the phenol- oxidase is suppressed completely by 10 -3 M sodium diethyldithi0carbamate, while mammalian peroxidase is resistant to the same concentration of sodium diethyldithio- carbamate (Okun, 1969). The cytochrome system activity is suppresed by means of formalin fixation (Barka and Anderson, 1963).

In our study, the use of formalin fixation suppressed Dopa oxidation through cytochrome system, while peroxidase activity can also be excluded because no reac- t:ion was seen in our preparations when adding 10 -3 sodium diethyldithiocarbamate. The observed oxidation process must, therefore, be ascribed only to the phenol oxidase activity.

It is possible that the small difference observed when comparing preparations incubated in solutions (1) and (2), especially in BDE animals, could be due to the fact that probably, even in the former solution, a certain degree of phenoloxidase activity was present during the incubation time.

2 1 0 H. Valenzuela et al.

b 30,um

C c 30 m

Fig. 2 a---c. Iris sections of an E3, fight brown, hooded rat (left vertical row) and of a BDE, black hoo&ed ra t (right vertical row) incubated at pH 7,4 for 90 rain: a preformed pigment in sections incubated in phosphate buffer alone; b sections incubated in phosphate buffer plus 5ram DL- Dopa show slight p igment formation in the E3 animal and a more pronounced one in the BDE rat; c disappearance o f induced pigment in sections incubated in phosphate buffer, 5ram DL- Dopa and 1 mM sodium diethyldithiocarbamate. N o counters tain

Phenol Oxidase Activity 211

Fig. 3 a--c. Ciliary body sections of an E3, light brown, hooded rat (left vertical row) and of a BDE, black hooded rat (right vertical row) incubated at pH 7.4 for 90 min: a preformed pig- men t in sections incubated in phosphate buffer alone; b sections incubated in phospha te buffer plus 5mM DL-Dopa show at the pigmented epithelium and s t roma a small a m o u n t o f newly formed pigment in tkte E3 animals and a more pronounced one in the BDE rats; c disappearance of induced pigment in sections incubated in phosphate buffer, 5 mM DL-Dopa and 1 mM sodium diethyldithiocarbamate. No counters ta in

212 H. Valenzuela et al.

References

Barka, T., Anderson, P.J.: Histochemistry. Hoeber Med. Div. p. 322, New York: Harper and Row 1963

Bloch, B.: Das Problem der Pigmentbildung in der Haut. Arch. Derm. Syph. 124, 129-208 (1917) Bloch, B. : Chemische Untersuchungen fiber das spezifische pigmentbildende Ferment der Haut,

die Dopa-oxidase. Z. physiol. Chem. 98, 226-254 (1917) Koch, H.R., Doldi, K.: Naphthalene cataracts in rats of differently pigmented strains. Exp. Eye

Res. 20, 180 (1975) Lillie, R.D.: Phenolic oxidative activities of the skin: some reactions of keratohyalin and trichohyalin

J. Histochem. Cytochem. 4, 318-330 (1956) Meirowsky, E. : A critical review of pigment research in the last hundred years. Brit. J. Derm. 52,

205-217 (1940) Miescher, G.: Die Pigmentgenese im Auge. Arch. mikr. Anat. 97, 326-396 (1923) Okun, M.R., Edelstein, L.M., Niebauer, G., Hamada, G.: The histochemical tyrosine-dopa reaction

for tyrosinase and its use in localizing tyrosinase activity in mast cells. J. Invest. Derm. 53, 39-45 (1969)

Received October 20, 1976