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Effect of degraded carrageenan on the intestine in germfree rats
Toxicology Letters, 8 (1981) 207-212 Elsevier/North-Holland
Press
EFFECT OF DEGRADED GERMFREE RATS
IWAO
HIRONO,
Department Tokyo,
207
Biomedical
YUKIKO
SUMI*,
of Carcinogenesis
Shirokanedai
CARRAGEENAN
KAZUNORI
ON THE INTESTINE
KUHARA
and MASASUMI
IN
MIYAKAWA*
and Cancer Susceptibiliry, Institute of Medical Science,
4-6-1, Minato-ku,
Tokyo 108, and *Laboratory
of Germfree
University of Life Research,
Nagoya University School of Medicine, Nagoya (Japan) (Received
January
19th, 1981)
(Accepted
January
26th,
1981)
SUMMARY
The role of intestinal by feeding 9 germfree days.
Animals
induced
essential
were sacrificed
by carrageenan
aggregates, extensive
bacterial
erosion,
flora in display
and 12 conventional
squamous
The germfree metaplasia
than those in the conventional for display
carrageenan
rats on diet containing
7, 20, 35, and 63 days after the start of feeding
were studied. and
of the effect of degraded
female Wistar
of the biological
rats showed
of the large
rats. Therefore,
effects
of degraded
mucosal
intestine,
it was concluded
and histological
lesions, and
was investigated
10% carrageenan
these
for 63 changes
such as macrophage lesions
that bacterial
were more flora
are not
carrageenan.
INTRODUCTION
Carrageenans, a heterogeneous various red seaweeds, are mainly
group of sulfated polygalactans extractable from K, h and 1 polymers, of which the last is readily
susceptible to acid hydrolysis, producing so-called ‘degraded’ carrageenan. Fabian et al. [l] observed when degraded carrageenan was administered to SpragueDawley rats, aggregation of macrophages in the lamina propria and submucosa of the distal rectum occurred first, followed by loss of mucosal glands, atrophy of the luminal epithelium, and replacement of the columnar epithelium by keratinizing stratified squamous epithelium. Furthermore, Wakabayashi et al. [7] reported induction of colorectal squamous cell carcinoma and adenocarcinoma in rats given degraded carrageenan. However, degraded carrageenan was not found to be mutagenic in Ames’ test (T. Matsushima, pets. commun.). Thus, it is unknown whether degraded carrageenan acts as a genotoxic carcinogen or an epigenetic carcinogen inducing acute and chronic inflammation of the mucous membrane of 0378.4274/81/0000~0000/$02.50
@I Elsevier/North-Holland
Biomedical
Press
the large intestine. inflammation Watt
of carrageenan
in induction
of intestinal
is also unknown.
and
parenterally
The mode of action
Marcus
[8]
did not induce
reported
that
degraded
ulcers in the large intestine
carrageenan of guinea
administered pigs. Therefore,
they concluded that degraded carrageenan given orally causes damage by a local action on the intestinal mucosa and they suggested that hydrolysis or fermentation of degraded carrageenan by bacteria may result in release of toxic products or that some alteration of the bacterial flora may induce changes in the large intestine. The final objective of our studies on carrageenan is to elucidate the mechanism of induction of intestinal tumors by degraded carrageenan. In this work using germfree rats, we examined whether the bacterial flora is involved in display of the biological effects
of carrageenan.
MATERIALS
AND
METHODS
Germfree and conventional female Wistar rats, bred as described previously [2] in the laboratory of Germfree Animal Research, Kawashima, Japan, were used. The germfree rats were maintained in a plastic isolator, and their germfree status was checked at the start and end of experiments and once a week during the experimental period as described by Wagner [6]. The degraded carrageenan used in this study was provided by Glaxo Laboratories, Paris, France; its sulfate content was about 30% and its average molecular weight was 20000-40000. Degraded carrageenan was added at a level of 10% to basal diet CL-2 prepared for germfree rats (CLEA Japan Inc., Tokyo). The basal diet CL-2 had the following composition, in parts per 100: crude carbohydrate, 48.2; crude protein, 23.7; ash, 8.9; crude cellulose, 5.6; crude fat, 5.6; and water, 8.0. The contents of vitamins A, D3, E, BI, Bz, and Bg were 2,000 and 400 I.U., and 41 .O, 3.4, 1.7, and 1.9 mg/lOO g diet, respectively. The content of vitamin Bt2 was 41.5 pg/lOO g diet. The main sources of crude carbohydrate were corn, wheat, and rice-bran; those of crude protein were fish meal and soybean cake; that of crude fat was soybean oil. The diet in polyethylene bags was irradiated with a dose of 5 megarad of gamma rays of Co-60. Diet sterilized in this way was confirmed to be sterile by repeated bacteriological examination. Nine germfree and 12 conventional rats of 48-52 days old were fed on sterilized 10% degraded carrageenan diet until the time of sacrifice. The food intakes per rat in the two groups were measured every 2 or 3 days. In germfree group, 3 rats were killed and autopsied on day 7, and 2 rats each on days 20, 35, and 63. In conventional group, 3 rats each were autopsied on the same schedule as in germfree group. Tissues were fixed in Carnoy’s fluid or 10% formalin, sectioned, and stained with H and E or toluidine blue. Histological examination of the intestine was performed using a Swiss-roll preparation.
209
RESULTS
The average days before
food intakes
per rat per day, determined
severe intestinal
lesions
appeared
by weight,
in the first 20
were 14.8 + 1.03 g in germfree
rats
and 15.5 + 0.61 g in conventional rats. Thus, there was no significant difference in the consumptions of degraded carrageenan by these two groups. After 2 weeks, blood was seen on the surface of the stools of germfree rats, and after 63 days the animals developed severe anemia. On the other hand, although blood was observed on the stools of conventional rats on day 6 of the experiment, it was not detected subsequently. Grossly, the large intestine from the anorectal junction to the distal colon of germfree rats developed a greyish white coating. This was particularly noticeable in germfree rats sacrificed on days 35 and 63. Only a slight coating was seen in conventional rats (Fig. 1). Histologically the coated area consisted of keratinized stratified squamous epithelium, which had replaced the glandular epithelium of the rectal and colonic
Fig. 1. Top: Gross carrageenan
appearance
for 63 days.
anorectal
junction
63 days.
Only a slight greyish
Arrows
in Figs.
of the colorectum
A greyish
to the distal colon.
white coating Bottom:
white coating
1 and 2 show the anorectal
of a germfree
rat fed diet containing
due to squamous-cell
Colorectum
of a conventional
was seen. The minimum junction.
metaplasia
10% degraded
was seen from the
rat fed on the same diet for
graduation
on the line is 2 mm.
710
I-ig. 2. Marked carrageenan
mucosa
squamous-cell
diet for 35 days.
(Fig.
metaplasia
of the colorectal
m~~cosa in a germfree
rat fed a degraded
H and E x 30.
2). Squamous
metaplasia
was
only
slight
in conventional
rats,
consistent with the gross appearance. Aggregates of large foamy macrophages containing material stained metachromatically with toluidine blue, suggesting the presence of carrageenan, in the lamina propria and submucosa of the colorectum and loss of mucosal glands were also more prominent in germfree rats than in conventional animals (Fig. 3). The extent of erosion was also more prominent in germfree rats than in conventional rats, except on day 7 of feeding when no significant difference was observed. Other peculiar findings were that germfree rats showed aggregates of macrophages and loss of mucosal glands in the cecum on day 35 and metachromatic material in the epithelium of the proximal convoluted tubules of the kidney on day 63. Material staining metachromatically with toluidine blue was more abundant in reticulum cells of the liver and mesenteric lymphnodes of germfree rats. Little metachromatic material was seen in the spleen of either germfree or conventional animals.
Fig. 3. Higher foamy
magnification
macrophages
of Fig. 2. In the lamina
propria
are seen. M, A part of squamous-cell
of the distal colon,
metaplasia.
Toluidine
aggregates
of large
blue x 340.
DISCUSSION
The present study showed that degraded carrageenan induced more severe mucosal lesions of the large intestine in germfree rats than in conventional animals. Thus, bacterial flora seem to be unnecessary for display of the biological effects of degraded carrageenan. Van Der Waaij et al. [5] reported that ulcerative lesions of the large intestine induced by degraded carrageenan in guinea pigs were significantly mitigated by selective elimination of aerobic Gram-negative intestinal microflora. Onderdonk et al. [4] reported that pretreatment with metronidazole, an antimicrobial primarily active against anaerobic bacteria, prevented carrageenaninduced colitis in guinea pigs. Furthermore, they [3] reported that germfree guinea pigs given degraded carrageenan did not develop cecal ulceration, but that animals associated with a conventional microflora acquired cecal ulcerations during carrageenan challenge. They inferred from these findings that carrageenan alone is not responsible for cecal ulceration and that bacteria are important in development of cecal ulcerations. In the present study, mucosal lesions associated with erosion of the large intestine induced by degraded carrageenan were much more extensive in germfree than in conventional rats. In observation of Onderdonk and Bartlett [3], the intestinal tissues from germfree quinea pigs given degraded carrageenan appeared to be entirely normal when compared to germfree animals not receiving carrageenan (A.B. Onderdonk, pers. commun.). The difference between their findings and ours on the biological effect of degraded carrageenan on germfree animals may be attributable to a species difference.
212
ACKNOWLEDGEMENT
This work Research
was supported
in part by a Grant-in-Aid
from the Ministry
of Education,
Science,
for Fundamental and Culture
Scientific
of Japan.
REFERENCES
R.J.
Fabian,
metaplasia
R.
M. Miyakawa, Germfree
Abraham,
of the rectal Studies
Research
A.B.
Onderdonk
Clin.
Nutr.,
A.B.
Onderdonk,
of rearing
germfree
and Gnotobiology,
and J.G.
J.A.
ulcerative
D. Van Der Waaij, guinea
pigs
Gastroenterology, M. Wagner,
Bartlett,
Hermes, colitis,
and
M.B.
Golberg,
Carrageenan-induced
Press, Cleveland,
Bacteriological
squamous
65 (1973) 265.
rats, in M. Miyakawa,
CRC
B. Cohen by
\electiw
J.L.
T.D.
Luckey
(Eds.),
Advances
in
1967, p. 48.
studies of experimental
ulcerative
colitis,
Am.
J.
and M. Anver, elimination
of germfree
T. Inagaki,
colorectal
in rats, (‘ancer
J. Watt and R. Marcus, 164.
and J.G.
Bartlett,
74 (IY78)
Mitigation of
the
Protective
of experimental
aerobic
effect
of metronidazole
in
521. inflammatory
Gram-negative
lntesrinal
bowel disease mlcroflora,
67 (1974) 460.
Determination
tumors
Dzink
Gastroenterology,
K. Wakabayashi,
12 (1971)
Coulston
in the rat, Gastroenterology,
32 (1979) 258.
experimental in
F.
mucosa
statu\,
Y. Fujimoto
1ett.,
Ann.
4 (1978)
Carrageenan-induced
N.Y.
and Y. Fukuda,
Acad.
Sci., 78 (1959) 89.
Induction
by degraded
carrageenan
171.
ulceration
of the large intestine
In the guinea
pig, (;LII,
of
Press
EFFECT OF DEGRADED GERMFREE RATS
IWAO
HIRONO,
Department Tokyo,
207
Biomedical
YUKIKO
SUMI*,
of Carcinogenesis
Shirokanedai
CARRAGEENAN
KAZUNORI
ON THE INTESTINE
KUHARA
and MASASUMI
IN
MIYAKAWA*
and Cancer Susceptibiliry, Institute of Medical Science,
4-6-1, Minato-ku,
Tokyo 108, and *Laboratory
of Germfree
University of Life Research,
Nagoya University School of Medicine, Nagoya (Japan) (Received
January
19th, 1981)
(Accepted
January
26th,
1981)
SUMMARY
The role of intestinal by feeding 9 germfree days.
Animals
induced
essential
were sacrificed
by carrageenan
aggregates, extensive
bacterial
erosion,
flora in display
and 12 conventional
squamous
The germfree metaplasia
than those in the conventional for display
carrageenan
rats on diet containing
7, 20, 35, and 63 days after the start of feeding
were studied. and
of the effect of degraded
female Wistar
of the biological
rats showed
of the large
rats. Therefore,
effects
of degraded
mucosal
intestine,
it was concluded
and histological
lesions, and
was investigated
10% carrageenan
these
for 63 changes
such as macrophage lesions
that bacterial
were more flora
are not
carrageenan.
INTRODUCTION
Carrageenans, a heterogeneous various red seaweeds, are mainly
group of sulfated polygalactans extractable from K, h and 1 polymers, of which the last is readily
susceptible to acid hydrolysis, producing so-called ‘degraded’ carrageenan. Fabian et al. [l] observed when degraded carrageenan was administered to SpragueDawley rats, aggregation of macrophages in the lamina propria and submucosa of the distal rectum occurred first, followed by loss of mucosal glands, atrophy of the luminal epithelium, and replacement of the columnar epithelium by keratinizing stratified squamous epithelium. Furthermore, Wakabayashi et al. [7] reported induction of colorectal squamous cell carcinoma and adenocarcinoma in rats given degraded carrageenan. However, degraded carrageenan was not found to be mutagenic in Ames’ test (T. Matsushima, pets. commun.). Thus, it is unknown whether degraded carrageenan acts as a genotoxic carcinogen or an epigenetic carcinogen inducing acute and chronic inflammation of the mucous membrane of 0378.4274/81/0000~0000/$02.50
@I Elsevier/North-Holland
Biomedical
Press
the large intestine. inflammation Watt
of carrageenan
in induction
of intestinal
is also unknown.
and
parenterally
The mode of action
Marcus
[8]
did not induce
reported
that
degraded
ulcers in the large intestine
carrageenan of guinea
administered pigs. Therefore,
they concluded that degraded carrageenan given orally causes damage by a local action on the intestinal mucosa and they suggested that hydrolysis or fermentation of degraded carrageenan by bacteria may result in release of toxic products or that some alteration of the bacterial flora may induce changes in the large intestine. The final objective of our studies on carrageenan is to elucidate the mechanism of induction of intestinal tumors by degraded carrageenan. In this work using germfree rats, we examined whether the bacterial flora is involved in display of the biological effects
of carrageenan.
MATERIALS
AND
METHODS
Germfree and conventional female Wistar rats, bred as described previously [2] in the laboratory of Germfree Animal Research, Kawashima, Japan, were used. The germfree rats were maintained in a plastic isolator, and their germfree status was checked at the start and end of experiments and once a week during the experimental period as described by Wagner [6]. The degraded carrageenan used in this study was provided by Glaxo Laboratories, Paris, France; its sulfate content was about 30% and its average molecular weight was 20000-40000. Degraded carrageenan was added at a level of 10% to basal diet CL-2 prepared for germfree rats (CLEA Japan Inc., Tokyo). The basal diet CL-2 had the following composition, in parts per 100: crude carbohydrate, 48.2; crude protein, 23.7; ash, 8.9; crude cellulose, 5.6; crude fat, 5.6; and water, 8.0. The contents of vitamins A, D3, E, BI, Bz, and Bg were 2,000 and 400 I.U., and 41 .O, 3.4, 1.7, and 1.9 mg/lOO g diet, respectively. The content of vitamin Bt2 was 41.5 pg/lOO g diet. The main sources of crude carbohydrate were corn, wheat, and rice-bran; those of crude protein were fish meal and soybean cake; that of crude fat was soybean oil. The diet in polyethylene bags was irradiated with a dose of 5 megarad of gamma rays of Co-60. Diet sterilized in this way was confirmed to be sterile by repeated bacteriological examination. Nine germfree and 12 conventional rats of 48-52 days old were fed on sterilized 10% degraded carrageenan diet until the time of sacrifice. The food intakes per rat in the two groups were measured every 2 or 3 days. In germfree group, 3 rats were killed and autopsied on day 7, and 2 rats each on days 20, 35, and 63. In conventional group, 3 rats each were autopsied on the same schedule as in germfree group. Tissues were fixed in Carnoy’s fluid or 10% formalin, sectioned, and stained with H and E or toluidine blue. Histological examination of the intestine was performed using a Swiss-roll preparation.
209
RESULTS
The average days before
food intakes
per rat per day, determined
severe intestinal
lesions
appeared
by weight,
in the first 20
were 14.8 + 1.03 g in germfree
rats
and 15.5 + 0.61 g in conventional rats. Thus, there was no significant difference in the consumptions of degraded carrageenan by these two groups. After 2 weeks, blood was seen on the surface of the stools of germfree rats, and after 63 days the animals developed severe anemia. On the other hand, although blood was observed on the stools of conventional rats on day 6 of the experiment, it was not detected subsequently. Grossly, the large intestine from the anorectal junction to the distal colon of germfree rats developed a greyish white coating. This was particularly noticeable in germfree rats sacrificed on days 35 and 63. Only a slight coating was seen in conventional rats (Fig. 1). Histologically the coated area consisted of keratinized stratified squamous epithelium, which had replaced the glandular epithelium of the rectal and colonic
Fig. 1. Top: Gross carrageenan
appearance
for 63 days.
anorectal
junction
63 days.
Only a slight greyish
Arrows
in Figs.
of the colorectum
A greyish
to the distal colon.
white coating Bottom:
white coating
1 and 2 show the anorectal
of a germfree
rat fed diet containing
due to squamous-cell
Colorectum
of a conventional
was seen. The minimum junction.
metaplasia
10% degraded
was seen from the
rat fed on the same diet for
graduation
on the line is 2 mm.
710
I-ig. 2. Marked carrageenan
mucosa
squamous-cell
diet for 35 days.
(Fig.
metaplasia
of the colorectal
m~~cosa in a germfree
rat fed a degraded
H and E x 30.
2). Squamous
metaplasia
was
only
slight
in conventional
rats,
consistent with the gross appearance. Aggregates of large foamy macrophages containing material stained metachromatically with toluidine blue, suggesting the presence of carrageenan, in the lamina propria and submucosa of the colorectum and loss of mucosal glands were also more prominent in germfree rats than in conventional animals (Fig. 3). The extent of erosion was also more prominent in germfree rats than in conventional rats, except on day 7 of feeding when no significant difference was observed. Other peculiar findings were that germfree rats showed aggregates of macrophages and loss of mucosal glands in the cecum on day 35 and metachromatic material in the epithelium of the proximal convoluted tubules of the kidney on day 63. Material staining metachromatically with toluidine blue was more abundant in reticulum cells of the liver and mesenteric lymphnodes of germfree rats. Little metachromatic material was seen in the spleen of either germfree or conventional animals.
Fig. 3. Higher foamy
magnification
macrophages
of Fig. 2. In the lamina
propria
are seen. M, A part of squamous-cell
of the distal colon,
metaplasia.
Toluidine
aggregates
of large
blue x 340.
DISCUSSION
The present study showed that degraded carrageenan induced more severe mucosal lesions of the large intestine in germfree rats than in conventional animals. Thus, bacterial flora seem to be unnecessary for display of the biological effects of degraded carrageenan. Van Der Waaij et al. [5] reported that ulcerative lesions of the large intestine induced by degraded carrageenan in guinea pigs were significantly mitigated by selective elimination of aerobic Gram-negative intestinal microflora. Onderdonk et al. [4] reported that pretreatment with metronidazole, an antimicrobial primarily active against anaerobic bacteria, prevented carrageenaninduced colitis in guinea pigs. Furthermore, they [3] reported that germfree guinea pigs given degraded carrageenan did not develop cecal ulceration, but that animals associated with a conventional microflora acquired cecal ulcerations during carrageenan challenge. They inferred from these findings that carrageenan alone is not responsible for cecal ulceration and that bacteria are important in development of cecal ulcerations. In the present study, mucosal lesions associated with erosion of the large intestine induced by degraded carrageenan were much more extensive in germfree than in conventional rats. In observation of Onderdonk and Bartlett [3], the intestinal tissues from germfree quinea pigs given degraded carrageenan appeared to be entirely normal when compared to germfree animals not receiving carrageenan (A.B. Onderdonk, pers. commun.). The difference between their findings and ours on the biological effect of degraded carrageenan on germfree animals may be attributable to a species difference.
212
ACKNOWLEDGEMENT
This work Research
was supported
in part by a Grant-in-Aid
from the Ministry
of Education,
Science,
for Fundamental and Culture
Scientific
of Japan.
REFERENCES
R.J.
Fabian,
metaplasia
R.
M. Miyakawa, Germfree
Abraham,
of the rectal Studies
Research
A.B.
Onderdonk
Clin.
Nutr.,
A.B.
Onderdonk,
of rearing
germfree
and Gnotobiology,
and J.G.
J.A.
ulcerative
D. Van Der Waaij, guinea
pigs
Gastroenterology, M. Wagner,
Bartlett,
Hermes, colitis,
and
M.B.
Golberg,
Carrageenan-induced
Press, Cleveland,
Bacteriological
squamous
65 (1973) 265.
rats, in M. Miyakawa,
CRC
B. Cohen by
\electiw
J.L.
T.D.
Luckey
(Eds.),
Advances
in
1967, p. 48.
studies of experimental
ulcerative
colitis,
Am.
J.
and M. Anver, elimination
of germfree
T. Inagaki,
colorectal
in rats, (‘ancer
J. Watt and R. Marcus, 164.
and J.G.
Bartlett,
74 (IY78)
Mitigation of
the
Protective
of experimental
aerobic
effect
of metronidazole
in
521. inflammatory
Gram-negative
lntesrinal
bowel disease mlcroflora,
67 (1974) 460.
Determination
tumors
Dzink
Gastroenterology,
K. Wakabayashi,
12 (1971)
Coulston
in the rat, Gastroenterology,
32 (1979) 258.
experimental in
F.
mucosa
statu\,
Y. Fujimoto
1ett.,
Ann.
4 (1978)
Carrageenan-induced
N.Y.
and Y. Fukuda,
Acad.
Sci., 78 (1959) 89.
Induction
by degraded
carrageenan
171.
ulceration
of the large intestine
In the guinea
pig, (;LII,
of