MELANOSIS COLI: STUDIES OF THE TOXIC EFFECTS OF IRRITANT PURGATIVES
H. W. STEERAND D. G. COLIN-JONES* Southampton University Medical School
PLATESCXII-CXIX
EXCESSIVE intake of irritant purgatives, such as the anthraquinones, is known to cause changes in certain tissues of the colon: the nerves, the smooth muscle and the macrophages (Smith, 1972). Anthraquinones are known to be potent cell poisons but previous studies have been concerned with either the production of pigmentation or the effects on the intrinsic innervation. Any common effect on the macrophages and the nerve cells has yet to be demonstrated. Macrophages become laden with pigment giving rise to the condition of melanosis coli. Much interest has been aroused as to the nature of this pigment which has been shown to have the staining characteristics of lipofuscin and not melanin (Pearse, 1972). The mode of formation of this pigment has yet to be elucidated. Under the influence of anthraquinones, the myenteric neurones initially enlarge but eventually there appears to be an atrophy and even a loss of some neurones with an increase in the number of Schwann cells in the ganglia (Smith, 1968, 1972). This study was undertaken in order to determine whether there is a similar cellular process affecting the different tissues. MATERIAL AND METHODS Rectal biopsies were obtained from the upper rectum with a Dick suction biopsy (Dick et al., 1970) at sigmoidoscopy. Specimens were obtained from seven patients aged 25 yr to 70 yr who were being investigated for functional gut disorders, in whom n o abnormality was found and who had not taken purgatives in the preceding 3 mth. Seven patients, aged 24 yr to 80 yr, suffering from melanosis coli were studied, (details are given in the table). Three patients (nos. 2, 6 and 7) were studied before and 3 to 7 mth after stopping taking the anthraquinone-containing purgatives. In addition, tissue was obtained from one patient who had only been taking anthraquinone-containing purgatives for 1 mth. Histochemistry The biopsies were immersed in 2-methylbutane and subsequently frozen in liquid nitrogen. Cryostat sections, 6 pm thick, were cut, placed on coverslips and air dried. All the sections were fixed in buffered 10per cent. neutral formalin at 4°C for 10min. except those used for the demonstration of /3-glucuronidase activity. These sections were fixed for 1hr in the formalin solution. The sections were then washed with distilled water and subjected to one of the following histochemical procedures. Received 16 Jan. 1974; accepted 10 June 1974.
* Present address; the Royal Hospital, Portsmouth J. PATH.-VOL.
115 (1975)
199
H. W. STEER AND D. G. COLIN-JONES
200
Lipofuscin was demonstrated by the Nile blue sulphate method or Schmorl's method (Pearse, 1972). A simultaneous coupling method was used for the identification of acid phosphatase (after Pearse, 1972) using naphthol AS-BI phosphate as the substrate and fast red violet LB as the diazonium salt. Non-specific esterases were demonstrated by a simultaneous coupling method (after Burstone, 1958) using either naphthol AS acetate or naphthol AS-D acetate as the substrate and fast blue BB as the diazonium salt. A post-coupling method was used for the identification of P-glucuronidase (after Fishman and Goldman, 1965) with naphthol AS-BI-PD glucuronide as the substrate and fast blue BB as the diazonium salt. Cholinesterases were demonstrated by the thiocholine method of Karnovsky and Roots (1964). TABLE The patients who had taken an excess of purgatives
Patient
__
-
Sex
Age
Drugs
Daily dose
Bowel action
Degree of melanosis
45 yr
0-5 times fluid
Very severe
50 yr
1-3 times soft
Severe
Duration
and stool
- 1. F.H.
2. M. S .
F
F
80
67
Senna
3 tabs.
Cascara Herbal remedies
2-3 tabs.
Cascara
2 tabs.
Senna
2 tabs.
3. E. H.
F
65
Cascara
5-14. tabs.
30 yr
1-6 times softlfluid
Severe
4. J. H.
F
73
Beechams pills (aloes)
2 tabs.
30+ yr
2-9 times
Severe
5 . M.H.
F
41
Dihydroxyanthraquinone Herbal remedies
Variable
?15 yr
1x /3 days
Moderate
6. K. H.
F
24
Cascara Brisacodyl
2 tabs.
15 yr
1x 14 days
Mild
7. V. T.
F
55
Senna Phenolphthalein
2 tabs.
6yr
1 x/3 days
Mild
-.
2 tabs. 2-3 tabs.
soft/fluid costive
costive
costive
~
Electron microscopy The biopsies were immediately fixed in cacodylate buffered 5 per cent glutaraldehyde (pH 7.3) for 4 hr at 4°C. They were subsequently washed in four changes of cacodylate buffered 10 per cent. sucrose solution for 24 hr and were then post-fixed in Verona1 acetate buffered 1 per cent. osmium tetroxide (pH 7.3) for 1 hr at 4"C, rinsed in chilled tap water and chilled 70 per cent. ethyl alcohol at 4"C, and dehydrated in a graded series of ethyl alcohol solutions ; propylene oxide was used as the clearing agent. The cleared tissues were embedded in Araldite. Sections 1 pm thick were cut with a glass knife on an ultramicrotome and stained by the method of Richardson, Jarrett and Fincke (1960). These sections were examined with a light microscope and, as a result, certain limited areas were selected from which sections approximately 25 nm were cut with a glass knife. These sections were stained with 1 per cent. uranyl acetate and Reynold's lead citrate, and examined in a Philips 300 electron microscope.
PURGATIVES AND MELANOSIS COLI
201
RESULTS
Histochemistry In normal colon (fig. 1) the macrophages are situated primarily near the luminal region of the tunica propria, whereas in the biopsy obtained from the patient who had taken anthraquinones for 1 mth (fig. 2), the macrophages were not only at the luminal border of the tunica propria but also near the basal region around the depths of the colonic glands. There was an increase in the macrophages at the latter site. No pigment with the staining characters of lipofuscin is detectable in this specimen. In melanosis coli there is a considerable increase in the number of macrophages in the tunica propria as well as an increase in their size (fig. 3). The macrophages are situated principally between the colonic glands, and in this region they can be divided into two groups: those near the luminal surface showing intense acid phosphatase (fig. 3) and non-specific esterase activity but no fl-glucuronidaseactivity, and those further from the lumen which show less intense acid phosphatase and non-specific esterase activity. There is a minimal amount of pigment in those macrophages in the region nearest to the lumen: most of the pigment is in those macrophages with less intense acid phosphatase activity (fig. 4). The more severe the degree of melanosis coli the larger is the pigmented zone in relation to the relatively non-pigmented region of macrophages. This pigment is stained by the Nile blue sulphate method and Schmorl’s method. In addition, it is periodic acid-Schiff positive, has a red fluorescence with fluorescent microscopy and is acid-fast. It, therefore, has the staining characteristics of lipofuscin. The neurones were located by their cholinesterase activity and serial sections were then subjected to other histochemical methods. The neurones of the submucosal plexus in melanosis coli have an increased acid phosphatase reaction (fig. 5 ) compared with those in normal colon, and they exhibit considerable non-specific esterase activity and some fl-glucuronidase activity. In melanosis coli the neurones occasionally have small lipofuscin granules. Within 3 mth of ceasing to take the anthraquinone-containing purgatives there is a decrease in the number of macrophages present and in the intensity of the acid phosphatase reaction (fig. 6). Ultrastructure The macrophages of the tunica propria in melanosis coli are not only more numerous and larger than those of normal colon, but also contain an increased number of lysosomes (fig. 7). These vary in size from small rounded electrondense granules of 0.3 pm diameter, to large irregular heterogeneously stained granules of up to 4 pm diameter. These latter are most abundant in the pigmented macrophages of light microscopy. All the lysosomal granules are membrane bound and have a characteristic membrane configuration (fig. 7), which differs from that of mitochondria. The lysosomal covering consists of an outer membrane, c. 100 8, thick separated by an electronlucent zone, c. 200 8,
202
H . W.STEER AND D . G . COLIN-JONES
wide, from an inner membrane, c. 100 8, thick. The inner membrane limits the dense matrix of the small lysosome but in the larger lysosomal bodies the inner membrane is infolded to varying degrees into this dense matrix (fig. 8) and is connected to bizarre membrane arrangements within these large lysosomes. The radiolucent zone widens at those sites where the inner membrane is infolded so that the lysosome appears to be bounded by a single membrane at these sites. The outer limiting membrane of the larger, heterogenous lysosomal bodies is frequently incomplete (fig. 8). A gradation can be seen from the small lysosomal granule to the large heterogeneous lysosomal bodies. The larger the lysosomal body the greater is the degree of infolding of the inner membrane. When the inner membrane is infolded that region of the lysosome in continuity with the radiolucent zone between the inner and outer membranes is less densely staining than the remainder of the lysosomal contents. The large heterogeneous lysosomal bodies also contain lipid aggregated into discrete droplets, and there are also lipid droplets unassociated with the lysosomal bodies. No intermediate forms were seen between the mitochondria and any of the lysosomal granules and no structures resembling degenerating mitochondria were detected in the lysosomal bodies. The mitochondria have an outer membrane, c. 70 A thick, separated by a radiolucent zone, c. 70 8, wide, from an inner membrane, c. 70 8,thick, which is infolded to form cristae mitochondriales (figs. 7 and 8). The neurones of the submucosal plexus in the normal colon contain occasional lysosomes, as well as mitochondria, and undistended rough and smooth-surfaced endoplasmic reticulum (fig. 9). In melanosis coli the neurones of the submucosal plexus contain an increased number of lysosomes (fig. 10). There are large lysosomal bodies in the perikaryon having heterogeneous contents and associated with lipid droplets (fig. 10). The mitochondria of these neurones appear to be increased and the rough-surfaced endoplasmic reticulum is distended (fig. 10). In the Schwann cells of the normal human colon there is a sparcity of cytoplasmic organelles (fig. 1l), but small lysosomal granules are occasionally seen. In melanosis coli the number of lysosomes in the Schwann cells is increased (fig. 12). These lysosomes are frequently larger than in the normal Schwann cell and are associated with lipid droplets (fig. 12). The membrane configuration of these iarger lysosomal bodies is identical to that of the lysosomes in the macrophages of patients with melanosis coli. DISCUSSION The relationship between irritant purgatives and melanosis coli was first propounded by Bartle (1928) and this association was substantiated by Bockus, Willard and Bank (1933). The mechanism of action of these purgatives, as well as the origin and nature of the pigment in the macrophages, has been the source of much study. The pigment is considered to be lipofuscin, a “wear and tear” pigment (Pearse, 1972). Lipofuscins have been studied in many tissues
PURGATIVES AND MELANOSIS COLI
203
and much is known about their relationship with certain cellular enzymes. Gomori (1955) found these pigments associated with non-specsc esterases. Gedigk and Bontke (1956) failed to demonstrate any p-glucuronidase activity associated with lipofuscins, but Goldfischer, Villaverde and Forschirm (1966) found both ,B-glucuronidase and acid phosphatase activity associated with lipofuscin in hepatocytes. Acid phosphatase and p-glucuronidase have been demonstrated in lysosomes (de Duve, 1959), and Holt (1963) found non-specific esterase activity in these cytoplasmic organelles. Ultrastructurally, Essner and Novikoff (1960) found acid phosphatase and a limiting membrane in hepatic lipofuscin granules. Thus, there is considerable histochemical and cytochemical evidence in favour of the lipofuscin being located within lysosomal particles. This association is confirmed by the present results in which acid hydrolases and lipofuscin have been found in macrophages, ganglion cells and Schwann cells. The presence of lysosomes and lysosome-like bodies in macrophages, ganglion cells and Schwann cells has been confirmed with the electron microscope. Intermediate forms between the typical small lysosomal bodies and large heterogeneous bodies have been observed. The largest heterogeneous bodies correlate with the ultrastructural description of Hibbs, Ferrens, Walsh and Burch (1965) for lipofuscin granules. Although the study of these authors was entirely ultrastructural, the present histochemical results, when correlated with the electron-microscopicfindings, confirm that the largest heterogeneous bodies are the lipofuscin-containing granules. Cytochemical tests are unable to differentiate between lysosomes and lipofuscin granules. Such a distinction would be misleading because the lipofuscin granules develop from lysosomal granules and intermediate forms would be expected. The absence of j3glucuronidase in the macrophages is not surprising: lysosomes in different tissues are known to vary in their content of acid hydrolases. The presence of lipofuscin and increased quantities of acid hydrolases in ganglion cells and Schwann cells is pertinent because of the findings of Wilkins and Hardcastle (1970). They showed that sennosides and oxanthrone act by stimulating colonic peristalsis. Whether this is a direct effect on the colonic nerve plexuses could not be elicited but Smith (1968, 1972) has demonstrated in man and experimentally in mice that the anthraquinone cathartics produce a morphological change in the myenteric plexus. There has been no evidence for the degeneration of mitochondria. No intermediate forms between mitochondria and lysosomes have been found. The membrane systems within the enlarged lysosomes are considered to be part of the intrinsic structure of lysosomes. The present results do not support the conclusion of Schrodt (1963) that the pigment originates from degenerating mitochondria. Wittoesch, Jackman and McDonald (1958) thought that the pigment was ingested and synthesised in the intestines but this view could not be substantiated by the present observations. The effect of anthraquinone cathartics in producing pigmentation and on the intrinsic innervation of the colon is the result of a common cytological response, involving the lysosomes, by different tissues (fig. 13). The
204
H. W. STEER AND D. G. COLIN-JONES
pathological effects of these drugs result from their influence on lysosomes in macrophages and nervous tissue. Anthraquinone Purgatives
I
1
Lysosomes
I .1
.I
Neurones Schwann cells
Macrophages
I
I
1
1
Loss of function
Pigmentation SUMMARY
Colonic biopsies from six patients with normal colons and seven patients with melanosis- coli were studied ultrastructurally and histochemically for lysosomal enzymes. Anthraquinone purgatives were found to increase the number of macrophages in the connective tissue of the colonic mucosa. In addition, they cause an increase in the intensity of lysosomal activity and an increase in the number of lysosomes in macrophages, Schwann cells and neurones of the submucosal plexus of the colonic mucosa. We gratefully acknowledge the facilities afforded to US by Professor D. Buliner, and the skilled technical assistance of Mr B. Backhouse and Mrs L. Baker. Mr B. J. Wilken kindly referred patient no. 5. REFERENCES BARTLE,H. J. 1928. The sigmoid: Anatomy, physiology, examhation and pathology. M . J. & Rec., 27, 521. BOCKUS,H. I., WILLARD,J. H., AND BANK,J. 1933. Melanosis coli: The etiologic significance of the anthracene laxatives: A report of 41 cases. J.A.M.A., 101, 1. M. S. 1958. The relationship between fixation and techniques for the histoBURSTONE, chemical localisation of hydrolytic enzymes. J. Histochem. Cytochem., 6, 322-339. DE DWE,C. 1959. Lysosomes, a new group of cytoplasmic particles. In SubcelluIar particles, edited by Teru Hayashi, The Ronald Press Company, New York, pp. 128-159. DICK,A. P., LENNARD-JONES, J. E., JONES, J. H., AND MORSON,B. C. 1970. Technique for suction biopsy of the rectal mucosa. Gut, 11, 182-184. ESSNER,E., AND NOVIKOFF, A. B. 1960. Human hepatocellular pigments and lysosomes. J. Ultrast. Res., 3, 374. FISHMAN, W. H., AND GOLDMAN, S. S. 1965. A postcoupling technique for glucuronidase employing the substrate, naphthol AS-BI-PD glucosiduronic acid. J. Histochem. Cytochern., 13,441-447. GEDIGK,P., AND BONTKE,E. 1956. Hydrolytic enzymes in lipopigments. Z . Zellforsch. mikrosk. Anat., 44, 495. GOLDFISCHER, S., VILLAVERDE, H., AND FORSCHIRM, R. 1966. The demonstration of acid hydrolase, thermostable reduced diphosphopyridine nucleotide tetrazolium reductase and peroxidase activities in human lipofuscin pigment granules. J. Histochem. Cytochem., 14, 641-652.
PURGATIVES NAD MELANOSIS COLI
205
GOMORI, G. 1955. Histochemistry of human esterases. J. Histochem. Cytochem., 3,479-484. HIBBS,R. G., FERRANS, V. J., WALSH,J. J., AND BURCH,G. E. 1965. Electron microscopic observations on lysosomes and related cytoplasmic components of normal and pathological cardiac muscle. Anat. Rec., 153, 173-186. HOLT,S. J. 1963. Some observations on the occurrence and nature of esterases in lysosomes. I n Lysosomes, edited by A. U. S. de Reuck and M. P. Cameron, J. and A. Churchill Ltd, London. KARNOYSKY, M. J., AND ROOTS,L. 1964. A " direct coloring " thiocholine method for cholinesterases. J. Histochem. Cytochem., 12, 219-221. PEARSE, A. G. E. 1972. Histochemistry. Theoretical and applied, 3rd ed., ChurchillLivingstone, Edinburgh and London. RICHARDSON, K. C., JAR RE^, L., AND FINICKE, E. H. 1960. Embedding in epoxy resin for ultrathin sectioning in electron microscopy. Stain Technol., 35, 313-323. SCHRODT, G. R. 1963. Melanosis coli: A study with the electron microscope. Dis. of Colon and Rectum, 6,277-283. SMITH, B. 1968. Effect of irritant purgatives on the myenteric plexus in man and the mouse. Gut, 9, 139-143. SMTH,B. 1972. Pathology of cathartic colon. Proc. roy. SOC.Med., 65, 288. WILKINS,J. L., AND HARDCASTLE, J. D. 1970. The mechanism by which senna glycosides and related compounds stimulate peristalsis in the human colon. Brit. J. Surg., 57, 864. J. H., JACKMAN, R. J,, AND MCDONALD, J. R. 1958. Melanosis coli: General WITTOESCH, review and a study of 887 cases. Dis. of Colon and Rectum, 1, 172-180.
STEERAND COLIN-JONES
PLATECXII PURGATIVES AND
FG 1.-Photomicrograph of the normal human colon. Acid phosphotase reaction.
FIG. 3.-Colonic
MELANOSIS COLI
FIG. 2.-Human colon, 1 mth after commencing the anthraquinone purgatives. Acid phosphatase reaction. Basal macrophages (M).
mucosa of a patient having melanosis coli. Acid phosphatase reaction.
PLATE CXIII
STEER AND COLIN-JONES PURGATIVES AND MELANOSIS
FIG.4.-Colonic mucosa of a patient having melanosis coli. Nile blue sulphate method. Luminal surface (L).
FIG. 6.-Colonic
COLI
FIG.5.-Submucosal neurones in the colon of a patient having melanosis coli. Acid phosphatase reaction.
mucosa of a patient having melanosis coiil 3 mth after stopping the anthraquinone ~urgatives. Acid phosphatase reaction.
PLATECXIV
STEERAND COLIX-JONES PURGATIVES AND MELANOSIS COLI
FIG.7.-Macrophage from the mucosa of a patient having melanosis coli. Nucleus (N). Lysosomes (LYS). Mitochondria (Mi).
STEERAND COLIN-JONES
PLATECXV
PURGATIVES AND
FIG. 8.-Macrophage
MELANOSIS COLI
from the mucosa of a patient having melanosis coli. Lysosomes (LYS). Mitochondria (Mi). Heterogeneous body (HB). Nucleus (N).
STEER AND
PLATECXVI
COLIN-JONES PURGATIVES AND MELANOSIS COLI
FIG.9.-Ganglion
cell (G) from the submucosal plexus of the normal human colon.
PLATECXVII
STEERAND COLIN-JONES PURGATIVES AND MELANOSIS
COLI
FIG.10.-Ganglion cell (G) from the submucosal plexus of the colon from a patient having melanosis coli. Lysosomes (LYS). Lipid (L). Distended rough surfaced endoplasmic reticulum (ER).
STEER AND COLIN-JONES
PLATE CXVIII PURGATIVES AND MELANOSIS COLI
FIG. 1I.-Schwann
cell (S) from the submucosal plexus of the normal human colon. Axons (A).
PLATECXIX
STEER AND COLIN-JONES
PURGATIVES AND MELANOSIS COLI
FIG. 12.--Schwann
cell (S) from the submucosal plexus of the colon from a patient having melanosis coii. Lysosomes (LYS). h o n s (A). Macrophage (M).
H. W. STEERAND D. G. COLIN-JONES* Southampton University Medical School
PLATESCXII-CXIX
EXCESSIVE intake of irritant purgatives, such as the anthraquinones, is known to cause changes in certain tissues of the colon: the nerves, the smooth muscle and the macrophages (Smith, 1972). Anthraquinones are known to be potent cell poisons but previous studies have been concerned with either the production of pigmentation or the effects on the intrinsic innervation. Any common effect on the macrophages and the nerve cells has yet to be demonstrated. Macrophages become laden with pigment giving rise to the condition of melanosis coli. Much interest has been aroused as to the nature of this pigment which has been shown to have the staining characteristics of lipofuscin and not melanin (Pearse, 1972). The mode of formation of this pigment has yet to be elucidated. Under the influence of anthraquinones, the myenteric neurones initially enlarge but eventually there appears to be an atrophy and even a loss of some neurones with an increase in the number of Schwann cells in the ganglia (Smith, 1968, 1972). This study was undertaken in order to determine whether there is a similar cellular process affecting the different tissues. MATERIAL AND METHODS Rectal biopsies were obtained from the upper rectum with a Dick suction biopsy (Dick et al., 1970) at sigmoidoscopy. Specimens were obtained from seven patients aged 25 yr to 70 yr who were being investigated for functional gut disorders, in whom n o abnormality was found and who had not taken purgatives in the preceding 3 mth. Seven patients, aged 24 yr to 80 yr, suffering from melanosis coli were studied, (details are given in the table). Three patients (nos. 2, 6 and 7) were studied before and 3 to 7 mth after stopping taking the anthraquinone-containing purgatives. In addition, tissue was obtained from one patient who had only been taking anthraquinone-containing purgatives for 1 mth. Histochemistry The biopsies were immersed in 2-methylbutane and subsequently frozen in liquid nitrogen. Cryostat sections, 6 pm thick, were cut, placed on coverslips and air dried. All the sections were fixed in buffered 10per cent. neutral formalin at 4°C for 10min. except those used for the demonstration of /3-glucuronidase activity. These sections were fixed for 1hr in the formalin solution. The sections were then washed with distilled water and subjected to one of the following histochemical procedures. Received 16 Jan. 1974; accepted 10 June 1974.
* Present address; the Royal Hospital, Portsmouth J. PATH.-VOL.
115 (1975)
199
H. W. STEER AND D. G. COLIN-JONES
200
Lipofuscin was demonstrated by the Nile blue sulphate method or Schmorl's method (Pearse, 1972). A simultaneous coupling method was used for the identification of acid phosphatase (after Pearse, 1972) using naphthol AS-BI phosphate as the substrate and fast red violet LB as the diazonium salt. Non-specific esterases were demonstrated by a simultaneous coupling method (after Burstone, 1958) using either naphthol AS acetate or naphthol AS-D acetate as the substrate and fast blue BB as the diazonium salt. A post-coupling method was used for the identification of P-glucuronidase (after Fishman and Goldman, 1965) with naphthol AS-BI-PD glucuronide as the substrate and fast blue BB as the diazonium salt. Cholinesterases were demonstrated by the thiocholine method of Karnovsky and Roots (1964). TABLE The patients who had taken an excess of purgatives
Patient
__
-
Sex
Age
Drugs
Daily dose
Bowel action
Degree of melanosis
45 yr
0-5 times fluid
Very severe
50 yr
1-3 times soft
Severe
Duration
and stool
- 1. F.H.
2. M. S .
F
F
80
67
Senna
3 tabs.
Cascara Herbal remedies
2-3 tabs.
Cascara
2 tabs.
Senna
2 tabs.
3. E. H.
F
65
Cascara
5-14. tabs.
30 yr
1-6 times softlfluid
Severe
4. J. H.
F
73
Beechams pills (aloes)
2 tabs.
30+ yr
2-9 times
Severe
5 . M.H.
F
41
Dihydroxyanthraquinone Herbal remedies
Variable
?15 yr
1x /3 days
Moderate
6. K. H.
F
24
Cascara Brisacodyl
2 tabs.
15 yr
1x 14 days
Mild
7. V. T.
F
55
Senna Phenolphthalein
2 tabs.
6yr
1 x/3 days
Mild
-.
2 tabs. 2-3 tabs.
soft/fluid costive
costive
costive
~
Electron microscopy The biopsies were immediately fixed in cacodylate buffered 5 per cent glutaraldehyde (pH 7.3) for 4 hr at 4°C. They were subsequently washed in four changes of cacodylate buffered 10 per cent. sucrose solution for 24 hr and were then post-fixed in Verona1 acetate buffered 1 per cent. osmium tetroxide (pH 7.3) for 1 hr at 4"C, rinsed in chilled tap water and chilled 70 per cent. ethyl alcohol at 4"C, and dehydrated in a graded series of ethyl alcohol solutions ; propylene oxide was used as the clearing agent. The cleared tissues were embedded in Araldite. Sections 1 pm thick were cut with a glass knife on an ultramicrotome and stained by the method of Richardson, Jarrett and Fincke (1960). These sections were examined with a light microscope and, as a result, certain limited areas were selected from which sections approximately 25 nm were cut with a glass knife. These sections were stained with 1 per cent. uranyl acetate and Reynold's lead citrate, and examined in a Philips 300 electron microscope.
PURGATIVES AND MELANOSIS COLI
201
RESULTS
Histochemistry In normal colon (fig. 1) the macrophages are situated primarily near the luminal region of the tunica propria, whereas in the biopsy obtained from the patient who had taken anthraquinones for 1 mth (fig. 2), the macrophages were not only at the luminal border of the tunica propria but also near the basal region around the depths of the colonic glands. There was an increase in the macrophages at the latter site. No pigment with the staining characters of lipofuscin is detectable in this specimen. In melanosis coli there is a considerable increase in the number of macrophages in the tunica propria as well as an increase in their size (fig. 3). The macrophages are situated principally between the colonic glands, and in this region they can be divided into two groups: those near the luminal surface showing intense acid phosphatase (fig. 3) and non-specific esterase activity but no fl-glucuronidaseactivity, and those further from the lumen which show less intense acid phosphatase and non-specific esterase activity. There is a minimal amount of pigment in those macrophages in the region nearest to the lumen: most of the pigment is in those macrophages with less intense acid phosphatase activity (fig. 4). The more severe the degree of melanosis coli the larger is the pigmented zone in relation to the relatively non-pigmented region of macrophages. This pigment is stained by the Nile blue sulphate method and Schmorl’s method. In addition, it is periodic acid-Schiff positive, has a red fluorescence with fluorescent microscopy and is acid-fast. It, therefore, has the staining characteristics of lipofuscin. The neurones were located by their cholinesterase activity and serial sections were then subjected to other histochemical methods. The neurones of the submucosal plexus in melanosis coli have an increased acid phosphatase reaction (fig. 5 ) compared with those in normal colon, and they exhibit considerable non-specific esterase activity and some fl-glucuronidase activity. In melanosis coli the neurones occasionally have small lipofuscin granules. Within 3 mth of ceasing to take the anthraquinone-containing purgatives there is a decrease in the number of macrophages present and in the intensity of the acid phosphatase reaction (fig. 6). Ultrastructure The macrophages of the tunica propria in melanosis coli are not only more numerous and larger than those of normal colon, but also contain an increased number of lysosomes (fig. 7). These vary in size from small rounded electrondense granules of 0.3 pm diameter, to large irregular heterogeneously stained granules of up to 4 pm diameter. These latter are most abundant in the pigmented macrophages of light microscopy. All the lysosomal granules are membrane bound and have a characteristic membrane configuration (fig. 7), which differs from that of mitochondria. The lysosomal covering consists of an outer membrane, c. 100 8, thick separated by an electronlucent zone, c. 200 8,
202
H . W.STEER AND D . G . COLIN-JONES
wide, from an inner membrane, c. 100 8, thick. The inner membrane limits the dense matrix of the small lysosome but in the larger lysosomal bodies the inner membrane is infolded to varying degrees into this dense matrix (fig. 8) and is connected to bizarre membrane arrangements within these large lysosomes. The radiolucent zone widens at those sites where the inner membrane is infolded so that the lysosome appears to be bounded by a single membrane at these sites. The outer limiting membrane of the larger, heterogenous lysosomal bodies is frequently incomplete (fig. 8). A gradation can be seen from the small lysosomal granule to the large heterogeneous lysosomal bodies. The larger the lysosomal body the greater is the degree of infolding of the inner membrane. When the inner membrane is infolded that region of the lysosome in continuity with the radiolucent zone between the inner and outer membranes is less densely staining than the remainder of the lysosomal contents. The large heterogeneous lysosomal bodies also contain lipid aggregated into discrete droplets, and there are also lipid droplets unassociated with the lysosomal bodies. No intermediate forms were seen between the mitochondria and any of the lysosomal granules and no structures resembling degenerating mitochondria were detected in the lysosomal bodies. The mitochondria have an outer membrane, c. 70 A thick, separated by a radiolucent zone, c. 70 8, wide, from an inner membrane, c. 70 8,thick, which is infolded to form cristae mitochondriales (figs. 7 and 8). The neurones of the submucosal plexus in the normal colon contain occasional lysosomes, as well as mitochondria, and undistended rough and smooth-surfaced endoplasmic reticulum (fig. 9). In melanosis coli the neurones of the submucosal plexus contain an increased number of lysosomes (fig. 10). There are large lysosomal bodies in the perikaryon having heterogeneous contents and associated with lipid droplets (fig. 10). The mitochondria of these neurones appear to be increased and the rough-surfaced endoplasmic reticulum is distended (fig. 10). In the Schwann cells of the normal human colon there is a sparcity of cytoplasmic organelles (fig. 1l), but small lysosomal granules are occasionally seen. In melanosis coli the number of lysosomes in the Schwann cells is increased (fig. 12). These lysosomes are frequently larger than in the normal Schwann cell and are associated with lipid droplets (fig. 12). The membrane configuration of these iarger lysosomal bodies is identical to that of the lysosomes in the macrophages of patients with melanosis coli. DISCUSSION The relationship between irritant purgatives and melanosis coli was first propounded by Bartle (1928) and this association was substantiated by Bockus, Willard and Bank (1933). The mechanism of action of these purgatives, as well as the origin and nature of the pigment in the macrophages, has been the source of much study. The pigment is considered to be lipofuscin, a “wear and tear” pigment (Pearse, 1972). Lipofuscins have been studied in many tissues
PURGATIVES AND MELANOSIS COLI
203
and much is known about their relationship with certain cellular enzymes. Gomori (1955) found these pigments associated with non-specsc esterases. Gedigk and Bontke (1956) failed to demonstrate any p-glucuronidase activity associated with lipofuscins, but Goldfischer, Villaverde and Forschirm (1966) found both ,B-glucuronidase and acid phosphatase activity associated with lipofuscin in hepatocytes. Acid phosphatase and p-glucuronidase have been demonstrated in lysosomes (de Duve, 1959), and Holt (1963) found non-specific esterase activity in these cytoplasmic organelles. Ultrastructurally, Essner and Novikoff (1960) found acid phosphatase and a limiting membrane in hepatic lipofuscin granules. Thus, there is considerable histochemical and cytochemical evidence in favour of the lipofuscin being located within lysosomal particles. This association is confirmed by the present results in which acid hydrolases and lipofuscin have been found in macrophages, ganglion cells and Schwann cells. The presence of lysosomes and lysosome-like bodies in macrophages, ganglion cells and Schwann cells has been confirmed with the electron microscope. Intermediate forms between the typical small lysosomal bodies and large heterogeneous bodies have been observed. The largest heterogeneous bodies correlate with the ultrastructural description of Hibbs, Ferrens, Walsh and Burch (1965) for lipofuscin granules. Although the study of these authors was entirely ultrastructural, the present histochemical results, when correlated with the electron-microscopicfindings, confirm that the largest heterogeneous bodies are the lipofuscin-containing granules. Cytochemical tests are unable to differentiate between lysosomes and lipofuscin granules. Such a distinction would be misleading because the lipofuscin granules develop from lysosomal granules and intermediate forms would be expected. The absence of j3glucuronidase in the macrophages is not surprising: lysosomes in different tissues are known to vary in their content of acid hydrolases. The presence of lipofuscin and increased quantities of acid hydrolases in ganglion cells and Schwann cells is pertinent because of the findings of Wilkins and Hardcastle (1970). They showed that sennosides and oxanthrone act by stimulating colonic peristalsis. Whether this is a direct effect on the colonic nerve plexuses could not be elicited but Smith (1968, 1972) has demonstrated in man and experimentally in mice that the anthraquinone cathartics produce a morphological change in the myenteric plexus. There has been no evidence for the degeneration of mitochondria. No intermediate forms between mitochondria and lysosomes have been found. The membrane systems within the enlarged lysosomes are considered to be part of the intrinsic structure of lysosomes. The present results do not support the conclusion of Schrodt (1963) that the pigment originates from degenerating mitochondria. Wittoesch, Jackman and McDonald (1958) thought that the pigment was ingested and synthesised in the intestines but this view could not be substantiated by the present observations. The effect of anthraquinone cathartics in producing pigmentation and on the intrinsic innervation of the colon is the result of a common cytological response, involving the lysosomes, by different tissues (fig. 13). The
204
H. W. STEER AND D. G. COLIN-JONES
pathological effects of these drugs result from their influence on lysosomes in macrophages and nervous tissue. Anthraquinone Purgatives
I
1
Lysosomes
I .1
.I
Neurones Schwann cells
Macrophages
I
I
1
1
Loss of function
Pigmentation SUMMARY
Colonic biopsies from six patients with normal colons and seven patients with melanosis- coli were studied ultrastructurally and histochemically for lysosomal enzymes. Anthraquinone purgatives were found to increase the number of macrophages in the connective tissue of the colonic mucosa. In addition, they cause an increase in the intensity of lysosomal activity and an increase in the number of lysosomes in macrophages, Schwann cells and neurones of the submucosal plexus of the colonic mucosa. We gratefully acknowledge the facilities afforded to US by Professor D. Buliner, and the skilled technical assistance of Mr B. Backhouse and Mrs L. Baker. Mr B. J. Wilken kindly referred patient no. 5. REFERENCES BARTLE,H. J. 1928. The sigmoid: Anatomy, physiology, examhation and pathology. M . J. & Rec., 27, 521. BOCKUS,H. I., WILLARD,J. H., AND BANK,J. 1933. Melanosis coli: The etiologic significance of the anthracene laxatives: A report of 41 cases. J.A.M.A., 101, 1. M. S. 1958. The relationship between fixation and techniques for the histoBURSTONE, chemical localisation of hydrolytic enzymes. J. Histochem. Cytochem., 6, 322-339. DE DWE,C. 1959. Lysosomes, a new group of cytoplasmic particles. In SubcelluIar particles, edited by Teru Hayashi, The Ronald Press Company, New York, pp. 128-159. DICK,A. P., LENNARD-JONES, J. E., JONES, J. H., AND MORSON,B. C. 1970. Technique for suction biopsy of the rectal mucosa. Gut, 11, 182-184. ESSNER,E., AND NOVIKOFF, A. B. 1960. Human hepatocellular pigments and lysosomes. J. Ultrast. Res., 3, 374. FISHMAN, W. H., AND GOLDMAN, S. S. 1965. A postcoupling technique for glucuronidase employing the substrate, naphthol AS-BI-PD glucosiduronic acid. J. Histochem. Cytochern., 13,441-447. GEDIGK,P., AND BONTKE,E. 1956. Hydrolytic enzymes in lipopigments. Z . Zellforsch. mikrosk. Anat., 44, 495. GOLDFISCHER, S., VILLAVERDE, H., AND FORSCHIRM, R. 1966. The demonstration of acid hydrolase, thermostable reduced diphosphopyridine nucleotide tetrazolium reductase and peroxidase activities in human lipofuscin pigment granules. J. Histochem. Cytochem., 14, 641-652.
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GOMORI, G. 1955. Histochemistry of human esterases. J. Histochem. Cytochem., 3,479-484. HIBBS,R. G., FERRANS, V. J., WALSH,J. J., AND BURCH,G. E. 1965. Electron microscopic observations on lysosomes and related cytoplasmic components of normal and pathological cardiac muscle. Anat. Rec., 153, 173-186. HOLT,S. J. 1963. Some observations on the occurrence and nature of esterases in lysosomes. I n Lysosomes, edited by A. U. S. de Reuck and M. P. Cameron, J. and A. Churchill Ltd, London. KARNOYSKY, M. J., AND ROOTS,L. 1964. A " direct coloring " thiocholine method for cholinesterases. J. Histochem. Cytochem., 12, 219-221. PEARSE, A. G. E. 1972. Histochemistry. Theoretical and applied, 3rd ed., ChurchillLivingstone, Edinburgh and London. RICHARDSON, K. C., JAR RE^, L., AND FINICKE, E. H. 1960. Embedding in epoxy resin for ultrathin sectioning in electron microscopy. Stain Technol., 35, 313-323. SCHRODT, G. R. 1963. Melanosis coli: A study with the electron microscope. Dis. of Colon and Rectum, 6,277-283. SMITH, B. 1968. Effect of irritant purgatives on the myenteric plexus in man and the mouse. Gut, 9, 139-143. SMTH,B. 1972. Pathology of cathartic colon. Proc. roy. SOC.Med., 65, 288. WILKINS,J. L., AND HARDCASTLE, J. D. 1970. The mechanism by which senna glycosides and related compounds stimulate peristalsis in the human colon. Brit. J. Surg., 57, 864. J. H., JACKMAN, R. J,, AND MCDONALD, J. R. 1958. Melanosis coli: General WITTOESCH, review and a study of 887 cases. Dis. of Colon and Rectum, 1, 172-180.
STEERAND COLIN-JONES
PLATECXII PURGATIVES AND
FG 1.-Photomicrograph of the normal human colon. Acid phosphotase reaction.
FIG. 3.-Colonic
MELANOSIS COLI
FIG. 2.-Human colon, 1 mth after commencing the anthraquinone purgatives. Acid phosphatase reaction. Basal macrophages (M).
mucosa of a patient having melanosis coli. Acid phosphatase reaction.
PLATE CXIII
STEER AND COLIN-JONES PURGATIVES AND MELANOSIS
FIG.4.-Colonic mucosa of a patient having melanosis coli. Nile blue sulphate method. Luminal surface (L).
FIG. 6.-Colonic
COLI
FIG.5.-Submucosal neurones in the colon of a patient having melanosis coli. Acid phosphatase reaction.
mucosa of a patient having melanosis coiil 3 mth after stopping the anthraquinone ~urgatives. Acid phosphatase reaction.
PLATECXIV
STEERAND COLIX-JONES PURGATIVES AND MELANOSIS COLI
FIG.7.-Macrophage from the mucosa of a patient having melanosis coli. Nucleus (N). Lysosomes (LYS). Mitochondria (Mi).
STEERAND COLIN-JONES
PLATECXV
PURGATIVES AND
FIG. 8.-Macrophage
MELANOSIS COLI
from the mucosa of a patient having melanosis coli. Lysosomes (LYS). Mitochondria (Mi). Heterogeneous body (HB). Nucleus (N).
STEER AND
PLATECXVI
COLIN-JONES PURGATIVES AND MELANOSIS COLI
FIG.9.-Ganglion
cell (G) from the submucosal plexus of the normal human colon.
PLATECXVII
STEERAND COLIN-JONES PURGATIVES AND MELANOSIS
COLI
FIG.10.-Ganglion cell (G) from the submucosal plexus of the colon from a patient having melanosis coli. Lysosomes (LYS). Lipid (L). Distended rough surfaced endoplasmic reticulum (ER).
STEER AND COLIN-JONES
PLATE CXVIII PURGATIVES AND MELANOSIS COLI
FIG. 1I.-Schwann
cell (S) from the submucosal plexus of the normal human colon. Axons (A).
PLATECXIX
STEER AND COLIN-JONES
PURGATIVES AND MELANOSIS COLI
FIG. 12.--Schwann
cell (S) from the submucosal plexus of the colon from a patient having melanosis coii. Lysosomes (LYS). h o n s (A). Macrophage (M).
