LOCALIZATION OF D E O X Y R I B O N U C L E A S E ACTIVITY BY THE SUBSTRATE FILM M E T H O D
1By R . D A O U S T2
Research Laboratories, Montreal Cancer Institute, Notre Dame Hospital and University of Montreal,
Montreal, Canada
I . I n t r o d u c t i o n . . . . 1 5 3 I I . T h e o r y o f t h e M e t h o d . . . 1 5 4 A . G e n e r a l C o n s i d e r a t i o n s . . . . 1 5 4 B . P r o p e r t i e s o f t h e S u b s t r a t e F i l m . . .. . . . 1 5 5
C. R e s o l u t i o n . . . . . . . . . . 1 5 5
I I I . I n s t r u m e n t a t i o n . . . . 1 5 7 A . P r e p a r a t i o n o f F r o z e n S e c t i o n s . . . . 1 5 7 B . M i c r o s c o p i c E x a m i n a t i o n o f D N A F i l m s . . . . . 1 5 8
C. P h o t o g r a p h y 1 5 8 I V . P r o c e d u r e . 1 5 8
A . P r e p a r a t i o n o f D N A F i l m s . . . 1 5 9 B . P r e p a r a t i o n o f T i s s u e s . . . \ 1 6 0 C . M o u n t i n g o f T i s s u e S e c t i o n s o n G e l a t i n e - G l y c e r o l S u p p o r t . . 1 6 0 D . E x p o s u r e o f D N A F i l m s t o T i s s u e S e c t i o n s . . . . . 1 6 0 E . S t a i n i n g o f D N A F i l m s a n d T i s s u e S e c t i o n s . . . . . 1 6 1 F . E x a m i n a t i o n a n d P h o t o g r a p h y o f D N A F i l m s a n d T i s s u e S e c t i o n s . 1 6 1
V . C r i t i q u e o f t h e M e t h o d 1 6 2 A . C o n t r o l S t u d i e s o n D N A F i l m s 1 6 2
B . R e s o l u t i o n 1 6 3 C. T i s s u e S e c t i o n s . . . . 1 6 4
D . C o m p a r i s o n o f H i s t o c h e m i c a l R e a c t i o n s f o r D N A a s e . . . 1 6 4
V I . R e s u l t s 1 6 6 V I I . A p p e n d i x : M a t e r i a l s a n d S o l u t i o n s . . . . 1 6 9
R e f e r e n c e s . . . . 1 6 9
I. INTRODUCTION
Deoxyribonuclease (DNAase) is an enzyme capable of hydrolysing deoxyribonucleic acid (DNA) to acid-soluble products by cleavage of phosphoric ester bonds between the nucleotide units of the molecule.
DNAases from various sources show many similar properties but may
1 I n v e s t i g a t i o n s s u p p o r t e d b y t h e N a t i o n a l C a n c e r I n s t i t u t e of C a n a d a .
2 R e s e a r c h A s s o c i a t e o f t h e N a t i o n a l C a n c e r I n s t i t u t e o f C a n a d a . 153
154 R. DAOUST
differ in their optimum p H of activity, their sensitivity to different acti
vators and inhibitors, the nature of the degradation products and their reaction to antisera (Laskowski, 1951, 1959; McDonald, 1955; Schmidt, 1955). DNAase will be used here as a general term, however, since the present article deals exclusively with the property of hydrolysing DNA referred to as DNAase activity.
DNA is known to play important roles in genetic mechanisms and growth processes (Brachet, 1957; Hotchkiss, 1955), and DNAase has received much attention because of its possible regulatory function in DNA metabolism. The enzyme has been the object of numerous bio
chemical investigations and shown to be widely distributed among tissues, but the biological significance of DNAase is still poorly under
stood. The experimental evidence to date suggests that its substrate, DNA, is a metabolically stable cell constituent (see discussion and refer
ences in Brachet,' 1957), and it appears that no particular intracellular function can be attributed to the DNAase present in the living cells. As histochemical analyses were expected to add significant information on this enzyme and possibly throw some light on its biological function(s), a method was devised for localizing DNAase activity in tissue sections by means of films of substrate (Daoust, 1957). The present paper deals with the theoretical aspects of the substrate film method and with tech
nical details on the localization of DNAase activity. A critique of the method is presented and the results obtained to date are reviewed.
II. THEORY OF THE METHOD A. GENERAL CONSIDERATIONS
The basis of the substrate film method is simple. A film of gelatine containing a substrate is placed in contact with tissue sections. During exposure, the substrate is transformed in the regions of the film covering the areas of tissue section containing the appropriate enzyme, and un- attacked in those parts overlying inactive areas. After exposure, either the unaltered substrate or the products of the reaction are visualized by staining. A reaction pattern or " a u t o g r a p h " is then observed in the film, and its comparison with the corresponding tissue sections reveals the sites of enzyme activity in the latter. For localizing DNAase activity, films composed of DNA dispersed in gelatine are used. After exposure to tissue sections, the DNA remaining in the film is stained with toluidine blue. The unstained regions of the film thus correspond to the areas of tissue sections possessing DNAase activity.
B . PROPERTIES OF THE SUBSTRATE F I L M
Several substrates can probably be included in gelatine films for studying enzyme activities of tissue sections. The staining method used to visualize the substrate remaining in the film after exposure does not need to be specific for that particular substrate. Any method can be utilized as long as the second component of the film, gelatine, does not stain by the same technique. Once satisfactory films of a given substrate have been prepared and a suitable staining reaction adopted, control studies must be carried out to ascertain that, under the conditions used with tissue sections, variations in the staining properties of the film are due exclusively to an enzymatic transformation of the substrate, While treatment with a solution of purified enzyme, for instance, should dupli
cate the results obtained with tissue sections, treatment with control solutions, on the other hand, should not alter the staining properties of the film. Similarly, no effect should be observed when films are exposed to inactivated tissue sections. Moreover, it must be ascertained, using con
trol gelatine films, that the disappearance of a substrate is not due to an effect on the supporting medium. The type and variety of control tests to be carried out will necessarily vary according to the nature of the substrate used and that of the enzymatic reaction investigated.
C. RESOLUTION
The localization of enzyme activity by the present method implies that a satisfactory contrast is obtained between stained and unstained regions of the film, and that relatively close negative areas can be dis
tinguished. Both the contrast and the resolution will depend on the concentration of the substrate in the film, the thickness of the film and the duration of the exposure—a situation in many ways similar to that encountered in radio-autography (Gross et al., 1951; Pelc, 1958).
1. Concentration of the Substrate in the Film
The concentration of the substrate in the film should be high enough to obtain a satisfactory contrast between the positive and negative regions of the films. In practice, films of a given thickness, but containing a substrate at different concentrations, are placed in contact with tissue sections at room temperature for various time intervals and, after separ
ation and staining, are examined under the microscope. The films con
taining the substrate at a concentration sufficient to give a satisfactory contrast after a relatively short exposure time (10-20 min) are judged the most convenient and similar films are used in further studies.
156 R . D A O U S T
2. Thickness of the Film and Exposure Time
The thickness of the film and the exposure time are probably the main factors affecting the resolution of the method. In a film placed in contact with a specimen containing point sources of enzyme activity (Fig. 1), the enzyme presumably diffuses in all directions above the contact line, and the unstained region corresponding to each point source will represent
— 2 t -
111!
POINT
SOURCE POINT
SOURCE
FILM
SPECIMEN
F I G . 1. S c h e m a t i c r e p r e s e n t a t i o n of s u b s t r a t e film a n d s o u r c e s of e n z y m e a c t i v i t y in s p e c i m e n for c a l c u l a t i o n of r e s o l v i n g p o w e r .
half a sphere the centre of which is at the point source. If the reaction is permitted to go on so that the enzyme diffuses throughout the film, a maximum contrast will be achieved between the positive and the negative regions. In such conditions, each reaction zone will have a radius equal to the thickness of the film (t)9 and the resolution will be equal to or less than 2t. I t is probably not necessary, however, to let the reaction proceed until the enzyme diffuses throughout the^film. If the reaction is stopped before, the contrast will be submaximal but may still be satisfactory and permit a better definition. Moreover, the reaction zones may probably overlap to some extent and still be distinguishable from each other. Thus, it is expected that the resolution of the method will be less than 2t in conditions of optimum visualization, and the thinner the film, the better will be the resolving power.
The exposure time should be optimum since too short an exposure would result in a poor contrast between the positive and negative regions of the film, while a prolonged exposure would result in a progressive loss in resolution. In practice, films of a minimum thickness, but containing a sufficient amount of substrate to obtain a satisfactory contrast, are exposed to tissue sections for various time intervals in each series of experiments. The films are then compared under the microscope, and those showing the optimum qualities with respect to contrast and resolu
tion are used for studying the distribution of enzyme activity.
3. Other Factors
Factors other than those discussed above may also have an effect on the resolution of the method. I t was observed, for instance, that the composition of the semi-solid support for sections is important in this respect, and that the addition of glycerol to the support improved the precision of the autographic image, presumably by affecting the rate of enzyme diffusion.
As far as the contact between the tissue sections and the film is con
cerned, the two elements must actually be in close contact so as to permit a diffusion of the enzyme in the film. If, in some regions, an interspace is left between the sections and the film, no diffusion occurs and no reac
tion is observed in the film over that area. However, such negative regions can easily be distinguished from those associated with inactive areas of tissue sections, as they do not correspond to particular histo
logical structures.
III. INSTRUMENTATION
The special instrumentation required for the substrate film method is limited to a microtome, a refrigerated cabinet, an oven at 45°C, a micro
scope and equipment for microphotography.
A. PREPARATION OF FROZEN SECTIONS
A microtome placed in a refrigerated cabinet is used in this laboratory for preparing sections of fresh frozen tissue. This method was found to present many advantages over the use of ordinary freezing microtomes at room temperature : (a) the tissues are kept at a constant and optimum temperature for sectioning, (b) the sections can be handled in a leisurely manner for their transfer from the microtome knife to the glass slide, and (c) the sections deposited on the slide are kept at low temperature while other sections are being prepared and transferred to the slide.
Two different types of refrigerated cabinets were used in this work, and satisfactory results were obtained with both types of cryostats. The first one, made according to Linderstrom-Lang and Mogensen (1938), consisted of an insulated wooden box cooled with dry ice and equipped with gloved armholes, an observation window and lighting (see also Glick,
1949). The cabinet now in use, similar to the one described by Coons et al., (1951), is cooled by mechanical refrigeration. The first type has the advantage of being cheaper but it is rather difficult with dry ice to keep the temperature within the optimum range, i.e. between —12 and — 20° C, for long periods. The second type is more convenient because it permits a better control of the "temperature and does not
158 R. DAOUST
require the regular addition of dry ice. I t is more expensive to build but, on the other hand, its operation cost is lower.
Any good microtome, of either hand sliding or rotary type, can be used in the cryostat. An important feature about the microtome,arrange- ment is to fit the knife with a device which prevents the curling of sections.
This device consists essentially of a glass "window" lying flat on the knife, but leaving a clearance of 50 μ between the guide and the blade.
The device was originally presented by Linderstrom-Lang and Mogen- sen (1938) and a modified model was proposed by Coons et al. (1951).
Β. MICROSCOPIC EXAMINATION OF DNA FILMS
The reaction patterns left in DNA films after contact with tissue sections and staining with toluidine blue are examined at low magni
fication (about 45 χ ) using a standard binocular research microscope. A yellow filter is placed in the light path to increase the contrast between the negative and positive regions of the film and, thus, facilitate the observation.
C. PHOTOGRAPHY
For photographing the reaction patterns in DNA films and the corre
sponding tissue sections, the slides are mounted on a monocular micro
scope fitted with a camera. The intensity of the light source is measured with a photometer, and adjusted to a constant reading before each series of experiments. A yellow filter is placed in the light path to increase the contrast between the clear and blue regions of DNA films. To focus sharply the image on the ground glass plate of the camera chamber, advantage is sometimes taken of the adhesion of fine particles to the DNA films after their contact with tissue sections. Final focusing is made by examining these particles with a focusing glass. High contrast films are used for photographing the reaction patterns in DNA films. The photography of tissue sections, on the other hand, presents no special problem.
IV. PROCEDURE
The essential steps of the substrate film method for localizing deoxy- ribonuclease activity are illustrated in Fig. 2 . Step 2a shows the materials used in their relative positions immediately before exposing the DNA film to tissue sections. Step 2b represents a cross-section of the same ele
ments during exposure. Step 2c shows the DNA film and the tissue sec
tions after separation and staining. Negative areas in the stained film correspond to the tissue sections.
A . PREPARATION OF D N A FILMS
A 1:1 mixture of gelatine 5% and D N A 0-2% is liquified by heating in a water bath. One drop (about 0 · 05 ml) is applied on a glass slide and rapidly spread over a surface of 2 · 5 χ 4 cm with the tip of the pipette.
GLASS SLIDE
GELATINE-DNA FILM TISSUE SECTION GELATINE-GLYCEROL GLASS SLIDE
®
\ ^ GLASS SLIDE{ • • ^ GELATINE-DNA FILM TISSUE SECTION :;:;:;;;:;:;:;:;:;:::;:::::::-T:;^— GELATINE - GLYCEROL
j GLASS SLIDE
- G L A S S SLIDE STAINED DNA FILM -UNSTAINED AREA
CORRESPONDING TO TISSUE SECTION
STAINED TISSUE SECTION GELATINE-GLYCEROL GLASS SLIDE
F I G . 2. E s s e n t i a l s t e p s of t h e D N A film m e t h o d for localizing D N A a s e a c t i v i t y in t i s s u e s e c t i o n s : (a, t o p ) , m a t e r i a l s u s e d i n t h e i r r e l a t i v e p o s i t i o n s before e x p o s i n g t h e D N A film t o t i s s u e s e c t i o n s ; (6, c e n t r e ) , cross-section of t h e s a m e e l e m e n t s d u r i n g e x p o s u r e ; (c, b o t t o m ) , D N A film a n d t i s s u e s e c t i o n s after s e p a r a t i o n a n d s t a i n i n g . T h e n e g a t i v e a r e a s in t h e s t a i n e d film c o r r e s p o n d t o t h e t i s s u e sections.
While the gelatine-DNA mixture is still fluid, the slide is placed on a level surface (room temperature) to insure uniformity of the film. The mixture then forms a gel and is allowed to dry completely. The film is fixed by standing overnight in neutral 1 0 ^ formaldehyde to render
160 R. DAOUST
DNA insoluble in water. The fixed gelatine-DNA film is then washed in three successive baths of distilled water, 15 min each, and allowed to dry again.
B. PREPARATION OF TISSUES
Under ether anaesthesia, the animal is exsanguinated from the ab
dominal aorta. The organs are excised with scissors, placed in a beaker kept on ice, washed in distilled water and blotted on filter paper. Pieces of organs are cut out with a scalpel, and held with forceps on a layer of ice covering a freezing stage which stands in crushed dry ice. When the pieces are completely frozen, water is added around their base with a medicine dropper in order to fix the pieces firmly to the freezing stage.
The stage is adjusted on the freezing microtome kept in a refrigerated cabinet at — 15°C and sufficient time is allowed for the tissues to adjust to that temperature. The tops of the pieces are then levelled off using first a scalpel and finally the microtome knife.
C. MOUNTING OF TISSUE SECTIONS ON GELATINE-GLYCEROL SUPPORT
When tissue sections are ready to be cut, a mixture of gelatine and glycerol (Baker, 1945; final concentrations used: 7% and 40% respec
tively) is liquefied by heating in a water bath. Ten drops (0-3-0-5 ml) are applied on a glass slide and spread with the tip of the pipette over a surface of 2 · 5 χ 5 cm. The slide is left a few minutes on a level surface at room temperature until gelation occurs. The slide is then introduced into the refrigerated box and held in one hand. Frozen sections are cut at 15 μ.
The sections lying flat on the microtome knife are picked up gently with a fine brush and deposited on the slide covered with gelatine-glycerol.
When many sections have been placed on the semi-solid support, the slide is taken out of the box and placed in a horizontal position in an oven at 45-50° C until the gelatine-glycerol support liquefies and the sections float on its surface. The slide is then transferred to a level surface at room temperature and the gelatine-glycerol is allowed to form a firm gel (5-10 min). This procedure of allowing the support to liquefy and reset while carrying tissue sections prevents the latter from adhering to the gelatine-DNA film when the slides are separated after exposure.
D. EXPOSURE OF DNA FILMS TO TISSUE SECTIONS
The slide supporting the tissue sections is placed against a gelatine- DNA film and the two slides are held together by means of paper clips.
The semi-solid gelatine-glycerol layer serves as a cushion and insures a
close contact between the sections and the film. After standing at room temperature for a given time interval, the slides are separated again.
The gelatine-DNA film is washed in two baths of distilled water, 5 min each, and allowed to dry. The slide bearing the tissue sections is fixed by standing in neutral 10% formaldehyde overnight. I t is then washed in two successive baths of distilled water, 5 min each, and left aside until the gelatine-glycerol support has dried.
E . STAINING OF DNA FILMS AND TISSUE SECTIONS
The gelatine-DNA film is dipped into a 0-2% solution of toluidine blue for 5 min. The slide is then rinsed rapidly in distilled water to remove excess dye and washed in two baths of distilled water, 5 min each. The film is allowed to dry again at room temperature and mounted under a coverslip using Canada balsam. Control films (gelatine 2-5%) do not show any appreciable staining in the same conditions.
The slide carrying the tissue sections is dipped into 1:3 acetic alcohol for 5 min before staining with toluidine blue. This prevents staining of supporting layer which contains a higher amount of gelatine than the gelatine-DNA film. The slide is transferred directly from acetic alcohol to a 0 · 1% solution of toluidine blue. After 1-2 min in the latter solution, the slide is rinsed rapidly in distilled water and washed in two baths of distilled water, 5 min each. I t is then allowed to dry at room temperature and mounted by adding Canada balsam and covering with a coverslip.
F . EXAMINATION AND PHOTOGRAPHY OF DNA FILMS AND TISSUE SECTIONS
The DNA film is examined under a binocular microscope at low mag
nification (45 χ ). A yellow filter is placed in the light path to obtain a better contrast between the stained and unstained regions of the film.
The positive reactions (i.e. the toluidine blue negative patterns left in the film by the action of tissue sections) are examined for their clarity and intensity. The patterns which are judged of good quality are encircled by a mark with ink on the coverslip. The slide with tissue sections is placed in a reverse position on the stage of the microscope, side by side with the slide supporting the DNA film. By displacing the mechanical stage, the sections corresponding to the different reaction patterns can rapidly be brought in the field and identified. Following the first exam
ination of DNA films and tissue sections, the slides are transferred to a monocular microscope fitted with a camera and a yellow filter. Pictures are taken of the reaction patterns and of the corresponding tissue sections
6
162 R. DAOUST
which had been encircled. The photographs are then compared to deter
mine the sites of DNAase activity in the tissue, i.e. the parts of the tissue sections corresponding to unstained areas in the DNA film.
F I G . 3. D N A films t r e a t e d w i t h c o n t r o l o r D N A a s e s o l u t i o n s a n d s t a i n e d w i t h t o l u i d i n e blue.
V. CRITIQUE OF THE METHOD
A. CONTROL STUDIES ON DNA F I L M S
The first point to be verified in the present method is that variations in the staining properties of the films are due exclusively to an enzymatic transformation of the substrate. If, for instance, unfixed gelatine-DNA films are dipped in a water bath, the DNA and the gelatine support dis
solve and such films, therefore, would not be adequate for studying the specific effect of DNAase. Formaldehyde-fixed gelatine-DNA films, on the other hand, were found to remain intact in water and control solution, while a progressive loss of DNA was observed by standing in a DNAase solution (Fig. 3). Thus, the fixed DNA films appeared suitable for studies on DNAase activity and were applied to tissue sections. Exposure of films to tissue sections resulted in a loss of DNA in those regions in contact with tissue sections (Figs. 4 and 5). No reaction was observed in films exposed to inactivated tissues (90°C, 10 min). As far as the supporting medium is concerned, no appreciable changes were noticed in control gelatine films left in contact with tissue sections and stained by phloxine.
Moreover, as observed with DNAase solution, treatment of a gelatine- DNA film by blood serum leaves a toluidine blue-negative film on the glass slide, indicating that the gelatine support remains intact during exposure. I t was concluded from these tests that the negative images left
in the films exposed to tissue sections were due to an hydrolysis of the film DNA by the DNAase contained in the tissues.
B . RESOLUTION
Theoretical considerations lead to the conclusion that in films exposed to tissue sections for an optimum time, the resolution of the method
164 R. DAOUST
should be less than twice the thickness of the film. Films prepared accord
ing to the technique described above were found to be about 10 μ thick on the average, and the resolution was observed to be actually of the order of 10 μ, i.e. practically equal to the thickness of the film.
The resolution of the present method does not compare advantage
ously with that of other histochemical methods which can reveal the intracellular distribution of enzymes. A localization at that level can sometimes be achieved with DNA films in special cases where the cells present a particular arrangement but the method, in its present stage at least, is not generally applicable to the cellular level. Nevertheless, the approach has already a wide range of possible applications at the tissue level, such as the localization of DNAase activity in particular cell types, or extracellular fluids of various organs, either normal or pathological (Daoust, 1958, 1960). Further work is being carried out to improve the resolution of the method and hence widen the scope of the present approach.
C . TISSUE SECTIONS
Fresh tissue sections have been used so far in this work in order to investigate the DNAase activities of tissues in conditions as close as possible to those prevailing in vivo. However, fixed tissue sections could probably be used also and, in fact, the film method appears very con
venient for studying the effect of various fixatives on the activity and the distribution of enzymes in tissues. Moreover, if the distribution of a given enzyme activity is found to be the same in a particular tissue before and after fixation, it might be advantageous in such cases to use fixed tissue sections, since the normal architecture would be retained to a higher degree in the latter sections than in fresh preparations.
D. COMPARISON OF HISTOCHEMICAL REACTIONS FOR D N A A S E
Each approach to the histochemical localization of enzymes presents particular advantages, but very few enzymes have yet been investigated using more than one method. As histochemical techniques become available in increasing number, however, it should be more common in the future to compare the enzyme distributions revealed by different methods. This would permit a better assessment of the various approaches. With regard to DNAase, three different histochemical approaches have been applied so far to the enzyme: (1) the fluorescent antibody method which localizes enzymes as proteins by immunological reactions (Marshall, 1954), (2) the precipitation method which demon
strates enzyme activity by precipitating degradation products in a visible form on tissue sections (Aronson et al., 1958) and (3) the present
substrate film method which reveals sites of enzyme activity by exposing films of substrate to tissue sections.
The fluorescent antibodies react with the antigenic material con
tained in tissue sections and, when applied to enzymes, localize the latter as proteins, irrespective of their activity or inactivity. I t would thus be possible to determine whether inactive DNAase exclusively may be found in some locations, by comparing the results of the antigen-antibody reac
tion with those of methods localizing the active DNAase. The compari
son would also be an excellent means of controlling the methods, as all sites showing DNAase activity should in addition react with the labelled antibodies.
An attempt has been made recently to adapt a phosphate precipi
tation method to the localization of DNAase activity (Aronson et al., 1958). However, the results reported so far are only preliminary, and further work is required to distinguish between the staining reactions
F I G. 6. D N A film e x p o s e d t o s e c t i o n of t h e s m a l l i n t e s t i n e (F I G. 7) for 10 m i n . P o s i t i v e r e a c t i o n ( w h i t e p a t t e r n ) c o r r e s p o n d i n g t o t h e c r y p t a n d villi e p i t h e l i a a n d t h e l u m e n b e t w e e n t h e villi. M a g n i f i c a t i o n : χ 40. F I G. 8. D N A film e x p o s e d t o s e c t i o n of t h e t h y r o i d (F I G . 9) for 5 m i n . D N A a s e a c t i v i t y s h o w n b y t h e colloid m a t e r i a l while t h e follicular cells a r e r e l a t i v e l y i n a c t i v e . M a g n i f i c a t i o n : χ 4 0 .
166 R . D A O U S T
due to tissue DNAase and tissue phosphatases. Moreover, it must be deter
mined whether the procedure reveals the actual sites of enzyme activity in the tissue, as the precipitation technique presents several risks in this regard (Danielli, 1953, 1958; Gomori, 1952; Pearse, 1960). If a reliable precipitation method is finally worked out to localize DNAase activity, it would indeed be interesting to compare the results obtained by the film method with those of the precipitation technique. The former presumably demonstrates the DNAase acting at the p H of the cells, while the latter method offers the possibility of localizing selectively acid or alkaline DNAase by p H adjustment of the incubation medium and addition of activators or inhibitors. The comparison might thus reveal differences between the actual and potential DNAase activities of tissues.
VI. RESULTS
The DNA film method has been applied so far to only a limited num
ber of tissues, and the results to date merely represent a first step in the study of DNAase distributions in normal and pathological tissues. Evi
dently, any attempt at making general inferences from such results would be premature at this stage.
The normal tissues investigated were the small intestine, the pancreas and the thyroid of the rat (Daoust, 1957). In the small intestine, DNAase activity was observed mainly in the epithelial cells of both the villi and the crypts of Lieberkuhn (Figs. 6 and 7). Relatively weak reactions were given by the other tissues composing the intestinal wall : the lamina pro
pria, the submucosa and the muscle layer. The reaction associated with the villi epithelium clearly extended beyond the surface into the lumen.
In the crypts of Lieberkuhn, the reactive sites did not correspond to the whole areas of the glands but rather to their central portions. Presumably, the DNAase is contained in the excretory region of the goblet cells of
F I G . 1 0 . D N A film e x p o s e d t o s e c t i o n of n o r m a l liver (FIG. 1 1 ) for 5 m i n . P o s i t i v e r e a c t i o n c o r r e s p o n d i n g t o liver p a r e n c h y m a . Slight D N A a s e a c t i v i t y o b s e r v e d in t h e blood vessels (arrows). M a g n i f i c a t i o n : χ 3 0 . F I G . 1 2 . D N A film e x p o s e d t o s e c t i o n of c i r r h o t i c liver (FIG. 1 3 ) for 4 0 m i n . D N A a s e a c t i v i t y i n t h e h y p e r p l a s t i c p a r e n c h y m a l n o d u l e s a n d r e l a t i v e i n a c t i v i t y of t h e bile d u c t a n d c o n n e c t i v e t i s s u e t r a b e c u l a e . M a g n i f i c a t i o n : χ 3 0 . F I G . 1 4 . D N A film e x p o s e d t o s e c t i o n of c i r r h o t i c liver (FIG. 1 5 ) for 4 0 m i n . N o t e a b s e n c e of r e a c t i o n i n a r e a c o r r e s p o n d i n g t o a t y p e of p a r e n c h y m a l t i s s u e c o n s i d e r e d a s site of origin of liver t u m o u r . M a g n i f i c a t i o n : χ 3 0 . F I G . 1 6 . D N A film e x p o s e d t o s e c t i o n of n e o p l a s t i c liver (FIG. 1 7 ) for 3 0 m i n . R e l a t i v e i n a c t i v i t y of t u m o u r ( h e p a t o m a ) cells c o m p a r e d w i t h s u r r o u n d i n g n o n - t u m o r a l t i s s u e o n left. T h e slight D N A a s e a c t i v i t y o b s e r v e d i n t h e t u m o u r m a s s is a s s o c i a t e d w i t h t r a b e c u l a e of bile d u c t a n d c o n n e c t i v e t i s s u e e l e m e n t s . M a g n i f i c a t i o n : χ 3 0 .
168 R . D A O U S T
both the villi and the crypt epithelia, and is discharged into the lumen together with the mucus. In the pancreas, a positive reaction was given by the acinar cells. This suggests that the pancreatic DNAase which reacts to labelled antibodies (Marshall, 1954), or part at least of the pro
tein, represents the active form of the enzyme. The thyroid DNAase activity was found to be localized mainly in the colloid material, the different follicles presenting, however, differences in intensity (Figs. 8 and 9). The follicular epithelium was relatively inactive. Positive reac
tions were also observed among the interfollicular connective tissue, possibly associated with small blood vessels.
An interesting application of the DNA film method was the study of the DNAase distribution in cirrhotic and neoplastic rat liver (Daoust,
1960; Daoust and Cantero, 1959). The distribution pattern of DNAase activity which is relatively uniform in normal liver, corresponding to the parenchyma (Figs. 10 and 11), was found to become heterogeneous in cirrhotic and neoplastic livers. The hyperplastic parenchymal nodules of cirrhotic liver usually showed intense DNAase activities, but the trabe
culae composed of bile ducts and connective tissue were relatively inac
tive (Figs. 12 and 13), as were the cystic bile ducts. The hyperplastic nodules themselves did not give a uniform reaction. A peculiar feature observed in some of these nodules was that parts presented positive reac
tions while other regions showed negligible DNAase activity (Figs. 14 and 15). Such inactive regions corresponded to a type of parenchymal tissue which, on the basis of its architecture and staining properties, is often considered as the site of origin of tumours. The liver tumours (hepatomas) were relatively inactive compared with the surrounding, non-tumoral tissue (Figs. 16 and 17). The weak reactions observed in the tumour masses were associated with trabeculae of bile duct and connective tissue elements. The necrotic parenchymal tissue, on the other hand, showed very high DNAase activities in either cirrhotic or neoplastic livers. Thus, the histochemical analysis has revealed that many changes which had been overlooked by the biochemical analyses of tissue homogenates, take place in the DNAase activity of rat liver during azo dye carcinogenesis.
ACKNOWLEDGEMENTS
I wish to thank Dr .Norman Nadler from the Department of Anatomy, McGill University, for reading and criticizing the manuscript, and Dr. A.
Gantero, Director of the Research Laboratories, Montreal Cancer Insti
tute, for his continued interest in the present studies. Part of the work described here v^as carried out at the Chester Beatty Research Institute in London, and Γ am much grateful to Professor A. Haddow for his co
operation and interest.
LOCALIZATION OF DEOXYRIBONUCLEASE ACTIVITY 169 VII. APPENDIX
MATERIALS AND SOLUTIONS
Gelatine-DNA. 20 mg of highly polymerized DNA are dissolved in 10 ml of distilled water, 24-48 hr being allowed for the solution to be effec
ted. In a second test tube, 0 · 5 g of gelatine (commercial sheet form) is dissolved in 10 ml of distilled water by heating in a water bath. The two solutions are then mixed and filtered through coarse filter paper.
The filtrate, 1:1 mixture of gelatine 5% and DNA 0 · 2%, is allowed to form a gel by standing at room temperature and is stored in a refrig
erator at 0-5° C.
Gelatine-glycerol. 1 · 4 g of gelatine is dissolved in 12 ml of distilled water, by heating in a water bath, and mixed with 8 ml of glycerol. The mix
ture is left at room temperature until gelation occurs and is stored in the refrigerator at 0-5° C.
Neutral 10% formaldehyde. 250 ml of stock solution (40% formaldehyde) are diluted to 11. with distilled water. The solution is neutralized by adding calcium carbonate in excess.
/ : 3 acetic-alcohol. 200 ml of glacial acetic acid are mixed with 600 ml of absolute ethyl alcohol.
Toluidine-blue solutions. 0-5 g of toluidine blue (for 0 - 1 % solution) or 1 g (for 0-2% solution) are dissolved in 500 ml of distilled water.
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