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Experimental Bile Duct Hyperplasia in Ducklings
Experimental Bile Duct Hyperplasia in Ducklings R.
0.
VLES
Unilever Research Laboratory, Vlaardingen, The Netherlands (Received for publication October 25, 1966)
case, it would be advisable to carry out a HE biological effects of aflatoxins, duckling test first or substitute it for the the toxic factors isolated from ground- time-consuming current investigations with nuts contaminated with some strains of As- rats and mice. In order to confirm the susceptibility of pergillus flavus, have been described extenday-old ducklings to liver carcinogens other sively (Wogan, 196S). than aflatoxin, compounds which are Asplin and Carnaghan (1961) showed known to induce liver tumours in rats, viz. that day-old ducklings are highly susceptiN;N-dimethylnitrosamine, thioacetamide, ble to aflatoxins; they found that these aniDL-ethionine, 2-acetylaminofluorene, 4-dimals are eminently suitable for screening methylaminoazobenzene (butter yellow) suspected samples of groundnut meal and for other experimental work connected with and 3'-methyl-4-dimethylaminoazobenzene this type of toxin: aflatoxins caused exten- and have been administered to ducklings. Furthermore, the effects of two bile sive bile duct proliferation in ducklings, acids—lithocholic and chenodeoxycholic even within a few days. Newberne et al. acid—on bile duct hyperplasia in ducklings (1964b) stated that anatoxin Bi, the have been investigated since it has been major toxic metabolite, administered in 5 shown that lithocholic acid induces biliary daily doses of 0.4 [Jig., initiated clearly proliferations in a wide variety of experidenned bile duct hyperplasia. The extent of this hyperplasia appeared to be related to mental animals (Eyssen et al., 1965; Leveille and Fairchild, 1965; Carey and Wilthe dose of anatoxin (Chang et al., 1963). liams, 1965). Various investigators have demonstrated EXPERIMENTAL that aflatoxin-containing diets led to the development of liver tumours in rats (LanGroups consisting of 4 to 9 one-day-old caster et al., 1961; Le Breton et al., 1962; Pekin ducklings were obtained from a comNewberne et al., 1964a), ducklings (Car- mercial hatchery and kept in heated cages naghan, 1965) and trout (Ashley et al., on wire floors. The animals received mash 1965). Local sarcomas have been produced (diets 1 and 2) or pellets (diet 3) and water by repeated subcutaneous injections of ad libitum. Diet 1 was a modified "Sheranatoxins in rats (Dickens and Jones, man-diet" consisting of 53% whole ground 1963) while carcinoma of the glandular wheat, 27% whole milk powder, 20% grass stomach have been observed in the same meal, salts and vitamins. Diet 2 (low-proanimal by Butler and Barnes (1966) who tein diet) consisted of 89.2% corn starch, believe that anatoxin is the most active 3.8% lard, 3.6% casein, 3.0% salt mixture carcinogenic substance known (Butler and and 0.4% vitamin mix. Diet 3 consisted of Barnes, 1963). pellets "Peking I" (J. L. van NieuwenhuIt has been suggested that the duckling izen's Graanhandel en Oliefabrieken N.V., could be a suitable experimental animal for The Netherlands). testing hepatotoxic compounds in general Feeding experiments and dosing experiand liver carcinogens in particular. In that ments were carried out using different INTRODUCTION
T
1368
1369
BILE D U C T HYPERPLASIA
levels of the following substances: 4-dimethylaminoazobenzene, 3'-methyl4-dimethylaminoazobenzene, DL-ethionine and dimethylnitrosamine (K & K Laboratories, Inc., Plainview, New York); thioacetamide (N.V. Koninklijke Pharmaceutische Fabrieken, v / h Brocades-Stheeman & Pharmacia, Amsterdam, The Netherlands) ; chenodeoxycholic acid, lithocholic acid and 2-acetylaminofluorene (KochLight Laboratories Ltd., Colnbrook-Bucks, England). Diet 1 was used in the feeding experiments. In the dosing experiments, the test compounds were either dissolved in water or suspended in 1,2-hydroxy-propane. The ducklings were daily intubated with 0.2 ml. of the solutions or suspensions by means of a polythene cannula attached to a hypodermic syringe. At the end of the experimental periods, the birds were decapitated, the livers were removed and samples were fixed in 10% neutral formalin. Paraffin sections were stained with Harris' hematoxylin-azophloxin and Masson's trichrome. The severity of the bile-duct proliferation was assessed in the liver sections using a scale from 0 to
4+. RESULTS The first photomicrogaph (Fig. 1) shows a normal portal tract in a control liver, t *
vl
:
" * " » . * i * -'•*'• -'-
•
Ji*
FIG. 2. Ductular cell and bile duct proliferation in liver of duckling intubated with IS ng. aflatoxin Bi over a S-day period, X 480. while in Fig. 2 the ductular cell reaction induced by aflatoxin (IS [Ag. over a 5-day period) is presented. As can be seen from Tables 1 and 2, 4-dimethylaminoazobenzene, 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene and chenodeoxycholic acid failed to induce any bile duct proliferation in the shortterm duckling test. Positive ductular cell reactions have been found after feeding DL-ethionine (Table 1 and Fig. 3), thioacetamide (Table 1 and Fig. 4 ) , lithocholic acid (Table 1 and Fig. 5), and after dosing dimethylnitrosamine (Table 2 and Fig. 6) and again lithocholic acid (Table 2). Dimethylnitrosamine induced the strongest reaction with extensive bile duct hyperplasia, proliferation of ductular cells, parenchymal necrosis and thickening of Glisson's capsule. DISCUSSION
* •
* • • *
•«> *'• / • * '
A.
*?»<*
« v
A
*
» • •
»
*
KM». #* 4 J • ?*fe*r* ,2-C * • ™ , » * ~ , FIG. 1. Portal tract in liver of control duckling. X 768.
The susceptibility of ducklings to several hepatotoxic substances has been demonstrated in these experiments. Hepatic lesions, designated as ductular cell reaction (Hunt et al., 1963) and consisting of proliferation of bile ductules and ducts, oval cells, collagen formation and leucocytic infiltration, have been induced in ducklings after administration of aflatoxin
1370
R. 0. VLES TABLE 1.—Feeding experiments Substance
Dose (%)
No. of ducklings
Days
2-Acetylaminofluorene
0 0.05 0.1 0.2
18 l.s 13 13
DL-Ethionine
0
13
0.025
Thioacetamide
2 1 2
0
1
13 2i,
n
21
o
2+
0.05
1 2 2
0 IS 26
0 2 0
o 1 1
0.1
5
13
5
0
0
3
26
0
0
0.025
1 2 2
0 13 26
(i
o
2 0
0
5 4
13 26
2 0
0.1 0.2
5 5
13 13
2 5
0.2
3 3
5 IS
0
1
(i
2+
3 3
5 IS
o 0
3+
0.05
Lithocholic acid
0.4
and several other structural unrelated substances. Butler (1964) observed biliary prolifera-
:
i*WM$&*\
+
2+ 2i
2+
+ + 2!
tion in the liver of ducklings after one single dose of dimethylnitrosamine. A comparable lesion was induced by cyasin, extract-
M k
S5
» • a -,
• ' • *
Bile duct hyperplasia
10 10 10
4-Dimethylaminoazobenzene
I *
Mortality
k
-•*
•'
T
FIG. 3. Ductular cell proliferation in liver of duckling fed 0.05% DL-ethionine in the diet during 13 days, x 768.
Fio. 4. Ductular cell proliferation in liver of duckling fed 0.05% thioacetamide in the diet during days 13 days, X 768.
1371
BILE DUCT HYPERPLASIA TABLE 2.—Dosing experiments
Diet
Vehicle
Dose (mg/0.2 mi.)
4-Dimethylaminoazobenzene
1
1, 2-Dihydroxypropane
0 7.2
3'-Methyl-4-dimethylaminoazobenzene
3
1,2-Dihydroxypropane
Substance
2
Dimethylnitrosamine
Lithocholic acid
Chenodeoxycholic acid
3
3
3
1,2-Dihydroxypropane
Water
1,2-Dihydroxypropane
1,2-Dihydroxypropane
ed from the cycad nut. The other substances tested by Butler (1964) namely carbon tetrachloride and the alkaloids, retrorsine and indicine, did not induce biliary proliferation. A single dose of ethionine or thioacetamide also failed to produce biliary proliferation. The present experiments, however, show that feeding 0.025% ethionine during 14 days, and 0.05% thioacetamide during 13
No. of ducklings
Days
Mortality
Bile duct hyperplasia
5 5
10 10
0 0
0 0
0
2 2
7 14
0 0
0 0
8
2 2
7 14
0 0
0 0
16
2 2
7 14
0 0
0 0
0
2 2
7 14
0 0
0 0
32
2 2
17 14
0 0
0 0
0.04 0.4
5 S
21 21
0 0
0
2
3 3 3
7 14 21
0 0 3
2+ 3+ 4+
4 8
9 9
7 7
5 9
3+ 3+
10
2 2
7 14
0 0
0 0
20
2 2
7 14
0 0
40
2 2
7 14
0 0
+ + + +
10
2 2
7 14
0 0
0 0
20
2 2
7 14
0 0
0 0
40
2 2
7 14
0 0
0 0
+
days induces a clearly denned biliary reaction. The pathology of dimethylnitrosamine poisoning in Pekin ducklings has been studied by Carlton et al. (1966). These authors stated that ducklings are more susceptible to the toxic effects of the drug than rats. Other known carcinogens, namely 1,2-3,4-diepoxybutane, l-oxo-4-nitroquinoline, (3-propionolactone and 4-dimethylami-
1372
R. 0 . VLES
FIG. 5. Bile duct and ductular cell proliferation in liver of duckling fed 0.4% lithocholic acid in the diet during 5 days, X 768.
noazobenzene have been fed to Pekin ducklings by the same authors. These drugs produced none or very few lesions and it was concluded that the duckling has no great advantage over other species as far as screening of potential carcinogens is concerned. Lithocholic acid produces bile duct proliferation in chickens, reptiles, rats, rabbits, mice, guinea pigs, hamsters and monkeys (Eyssen et al., 1965; Leveille and Fairchild, 1965; Carey and Williams, 1965). It is worth remembering here that the socalled "primary" bile acids (cholic and chenodeoxycholic acid) are formed directly from cholesterol in the liver (Anonymous, 1964). As shown in Fig. 7, the "secondary" bile acids (deoxycholic and lithocho-
lie acid) are formed from the "primary" acids by removal of the 7-hydroxy groups by intestinal bacteria. The duckling appears to be an experimental animal which is rather susceptible to the effects of lithocholic acid. Chenodeoxycholic acid produces no ductular cell reaction in ducklings, a result similar to that found in chickens by Leveille and Fairchild (1965). Popper and Schaffner (1961) suggested that the ductular cell reaction could be the result of excretion of an irritating material into the ductules where this material serves as a stimulant for ductular proliferation, inflammatory cell reaction and periductular fibrosis. Lithocholic acid may represent or induce the synthesis of such material. It is conceivable that the same mechanism is involved in the bile duct proliferations observed after administration of aflatoxin, DL-ethionine, thioacetamide or dimethylnitrosamine. SUMMARY
The susceptibility of ducklings to known hepatocarcinogenic substances in producing bile duct proliferation was investigated. Liver changes designated as "biliary ductular cell reaction" and defined as a proliferation of bile ductules and oval cells, collagen BILE
METABOLITES
ACIDS
intestine
liver
'OH CHOLIC ACID
DEOXYCHOLIC
ACID
UTHOCHCKIC
ACID
*
:• . : # . - • >'V;-FIG. 6. Bile duct proliferation in liver of duckling intubated daily with 4 mg. dimethylnitrosamine during 1 week, X 480.
CHENOOEOXYCHOIIC
ACID
FIG. 7. Removal of the 7-hydroxy groups from bile acids by intestinal bacteria.
BILE DUCT HYPERPLASIA
formation and leucocytic infiltration, have been induced in a short time, in ducklings, by DL-ethionine, thioacetamide, dimethylnitrosamine and lithocholic acid. The pathological significance of the ductular cell reaction as well as the possible role of some bile acids in its genesis are discussed. REFERENCES Anonymous, 1964. Lithocholic acid and biliary ductular cellular reaction in animals. Nutr. Rev. 22: 305-306. Ashley, L. M., J. E. Halver, W. K. Gardner and G. N. Wogan, 1965. Crystalline anatoxins cause trout hepatoma. Federation Proc. 24: 627. Asplin, F. D., and R. B. A. Carnaghan, 1961. The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Record, 73: 1215-1218. Butler, W. H., 1964. Acute liver injury in ducklings as a result of anatoxin poisoning. J. Pathol. Bacteriol. 88: 189-195. Butler, W. H., and J. M. Barnes, 1963. Toxic effects of groundnut meal containing aflatoxin to rats and guinea-pigs. Brit. J. Cancer, 17: 699-710. Butler, W. H., and J. M. Barnes, 1966. Carcinoma of the glandular stomach in rats given diets containing anatoxin. Nature, 209: 90. Carey, Jr., J. B., and G. Williams, 1965. Lithocholic acid in human blood serum. Science, 150: 620-622. Carlton, W. W., J. E. Lord and L. Friedman, 1966. Pathology of dimethylnitrosamine-poisoning in Pekin ducklings. Toxicol. Appl. Pharmacol. 8: 224-234. Carnaghan, R. B. A., 1965. Hepatic tumours in ducks fed a low level of toxic groundnut meal. Nature, 208: 308. Chang, S. B., M. M. Abdel Kader, E. L. Wick and G. N. Wogan, 1963. Anatoxin B 2 : chem-
1373
ical identity and biological activity. Science, 142:1191-1192. Dickens, F., and H. E. H. Jones, 1963. The carcinogenic action of aflatoxin after its subcutaneous injection in the rat. Brit. J. Cancer, 17 : 691-698. Eyssen, H., M. Vandeputte and E. Evrard, 1965. Effect of various dietary bile acids on nutrient absorption and on liver size in chicks. Arch. Inter. Pharmacodyn. 158: 292-306. Hunt, R. D., G. A. Leveille and H. E. Sauberlich, 1963. Dietary bile acids and lipid metabolism II. The ductular cell reaction induced by lithocholic acid. Proc. Soc. Exptl. Biol. Med. 113: 139-142. Lancaster, M. C , F. P. Jenkins and J. McL. Philp, 1961. Toxicity associated with certain samples of groundnuts. Nature, 192: 10951096. Le Breton, E., C. Frayssinet and J. Boy, 1962. Sur l'apparition d'hepatomes "spontanes" chez le rat Wistar. Role de la toxine de ['Aspergillus flavus. Interet en pathologic humaine et cancerologie experimentale. Compt. Rend. 255: 784-786. Leveille, G. A., and D. G. Fairchild, 1965. Lack of effect of dietary chenodeoxycholic acid on plasma and liver lipids and the ductular cell reaction in the chick. Poultry Sci. 44: 16111612. Newberne, P. M., W. W. Carlton and G. N. Wogan, 1964a. Hepatomas in rats and hepatorenal injury in ducklings fed peanut meal or Aspergillus flavus extract. Pathol. Vet. 1: 105-132. Newberne, P. M., G. N. Wogan, W. W. Carlton and M. M. Abdel Kader, 1964b. Histopathologic lesion in ducklings caused by Aspergillus flavus cultures, culture extracts and crystalline anatoxins. Toxicol. Appl. Pharmacol. 6: 542556. Popper, H., and F. Schaffner, 1961. Response of the Liver to Injury in Progress in Liver Diseases. Grune & Stratton, Inc., New York, p. 86. Wogan, G. N., 1965. Editor, Mycotoxins in Foodstuffs. The Massachusetts Institute of Technology Press.
NEWS AND NOTES (Continued from page 1367) from a scientific and practical point of view. An additional highlight will be the symposium on the "Exchange of knowledge in poultry produc-
tion between Europe and the developing countries." In this connection there will be an exhibition displaying the achievements of the European
(Continued on page 1397)
0.
VLES
Unilever Research Laboratory, Vlaardingen, The Netherlands (Received for publication October 25, 1966)
case, it would be advisable to carry out a HE biological effects of aflatoxins, duckling test first or substitute it for the the toxic factors isolated from ground- time-consuming current investigations with nuts contaminated with some strains of As- rats and mice. In order to confirm the susceptibility of pergillus flavus, have been described extenday-old ducklings to liver carcinogens other sively (Wogan, 196S). than aflatoxin, compounds which are Asplin and Carnaghan (1961) showed known to induce liver tumours in rats, viz. that day-old ducklings are highly susceptiN;N-dimethylnitrosamine, thioacetamide, ble to aflatoxins; they found that these aniDL-ethionine, 2-acetylaminofluorene, 4-dimals are eminently suitable for screening methylaminoazobenzene (butter yellow) suspected samples of groundnut meal and for other experimental work connected with and 3'-methyl-4-dimethylaminoazobenzene this type of toxin: aflatoxins caused exten- and have been administered to ducklings. Furthermore, the effects of two bile sive bile duct proliferation in ducklings, acids—lithocholic and chenodeoxycholic even within a few days. Newberne et al. acid—on bile duct hyperplasia in ducklings (1964b) stated that anatoxin Bi, the have been investigated since it has been major toxic metabolite, administered in 5 shown that lithocholic acid induces biliary daily doses of 0.4 [Jig., initiated clearly proliferations in a wide variety of experidenned bile duct hyperplasia. The extent of this hyperplasia appeared to be related to mental animals (Eyssen et al., 1965; Leveille and Fairchild, 1965; Carey and Wilthe dose of anatoxin (Chang et al., 1963). liams, 1965). Various investigators have demonstrated EXPERIMENTAL that aflatoxin-containing diets led to the development of liver tumours in rats (LanGroups consisting of 4 to 9 one-day-old caster et al., 1961; Le Breton et al., 1962; Pekin ducklings were obtained from a comNewberne et al., 1964a), ducklings (Car- mercial hatchery and kept in heated cages naghan, 1965) and trout (Ashley et al., on wire floors. The animals received mash 1965). Local sarcomas have been produced (diets 1 and 2) or pellets (diet 3) and water by repeated subcutaneous injections of ad libitum. Diet 1 was a modified "Sheranatoxins in rats (Dickens and Jones, man-diet" consisting of 53% whole ground 1963) while carcinoma of the glandular wheat, 27% whole milk powder, 20% grass stomach have been observed in the same meal, salts and vitamins. Diet 2 (low-proanimal by Butler and Barnes (1966) who tein diet) consisted of 89.2% corn starch, believe that anatoxin is the most active 3.8% lard, 3.6% casein, 3.0% salt mixture carcinogenic substance known (Butler and and 0.4% vitamin mix. Diet 3 consisted of Barnes, 1963). pellets "Peking I" (J. L. van NieuwenhuIt has been suggested that the duckling izen's Graanhandel en Oliefabrieken N.V., could be a suitable experimental animal for The Netherlands). testing hepatotoxic compounds in general Feeding experiments and dosing experiand liver carcinogens in particular. In that ments were carried out using different INTRODUCTION
T
1368
1369
BILE D U C T HYPERPLASIA
levels of the following substances: 4-dimethylaminoazobenzene, 3'-methyl4-dimethylaminoazobenzene, DL-ethionine and dimethylnitrosamine (K & K Laboratories, Inc., Plainview, New York); thioacetamide (N.V. Koninklijke Pharmaceutische Fabrieken, v / h Brocades-Stheeman & Pharmacia, Amsterdam, The Netherlands) ; chenodeoxycholic acid, lithocholic acid and 2-acetylaminofluorene (KochLight Laboratories Ltd., Colnbrook-Bucks, England). Diet 1 was used in the feeding experiments. In the dosing experiments, the test compounds were either dissolved in water or suspended in 1,2-hydroxy-propane. The ducklings were daily intubated with 0.2 ml. of the solutions or suspensions by means of a polythene cannula attached to a hypodermic syringe. At the end of the experimental periods, the birds were decapitated, the livers were removed and samples were fixed in 10% neutral formalin. Paraffin sections were stained with Harris' hematoxylin-azophloxin and Masson's trichrome. The severity of the bile-duct proliferation was assessed in the liver sections using a scale from 0 to
4+. RESULTS The first photomicrogaph (Fig. 1) shows a normal portal tract in a control liver, t *
vl
:
" * " » . * i * -'•*'• -'-
•
Ji*
FIG. 2. Ductular cell and bile duct proliferation in liver of duckling intubated with IS ng. aflatoxin Bi over a S-day period, X 480. while in Fig. 2 the ductular cell reaction induced by aflatoxin (IS [Ag. over a 5-day period) is presented. As can be seen from Tables 1 and 2, 4-dimethylaminoazobenzene, 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene and chenodeoxycholic acid failed to induce any bile duct proliferation in the shortterm duckling test. Positive ductular cell reactions have been found after feeding DL-ethionine (Table 1 and Fig. 3), thioacetamide (Table 1 and Fig. 4 ) , lithocholic acid (Table 1 and Fig. 5), and after dosing dimethylnitrosamine (Table 2 and Fig. 6) and again lithocholic acid (Table 2). Dimethylnitrosamine induced the strongest reaction with extensive bile duct hyperplasia, proliferation of ductular cells, parenchymal necrosis and thickening of Glisson's capsule. DISCUSSION
* •
* • • *
•«> *'• / • * '
A.
*?»<*
« v
A
*
» • •
»
*
KM». #* 4 J • ?*fe*r* ,2-C * • ™ , » * ~ , FIG. 1. Portal tract in liver of control duckling. X 768.
The susceptibility of ducklings to several hepatotoxic substances has been demonstrated in these experiments. Hepatic lesions, designated as ductular cell reaction (Hunt et al., 1963) and consisting of proliferation of bile ductules and ducts, oval cells, collagen formation and leucocytic infiltration, have been induced in ducklings after administration of aflatoxin
1370
R. 0. VLES TABLE 1.—Feeding experiments Substance
Dose (%)
No. of ducklings
Days
2-Acetylaminofluorene
0 0.05 0.1 0.2
18 l.s 13 13
DL-Ethionine
0
13
0.025
Thioacetamide
2 1 2
0
1
13 2i,
n
21
o
2+
0.05
1 2 2
0 IS 26
0 2 0
o 1 1
0.1
5
13
5
0
0
3
26
0
0
0.025
1 2 2
0 13 26
(i
o
2 0
0
5 4
13 26
2 0
0.1 0.2
5 5
13 13
2 5
0.2
3 3
5 IS
0
1
(i
2+
3 3
5 IS
o 0
3+
0.05
Lithocholic acid
0.4
and several other structural unrelated substances. Butler (1964) observed biliary prolifera-
:
i*WM$&*\
+
2+ 2i
2+
+ + 2!
tion in the liver of ducklings after one single dose of dimethylnitrosamine. A comparable lesion was induced by cyasin, extract-
M k
S5
» • a -,
• ' • *
Bile duct hyperplasia
10 10 10
4-Dimethylaminoazobenzene
I *
Mortality
k
-•*
•'
T
FIG. 3. Ductular cell proliferation in liver of duckling fed 0.05% DL-ethionine in the diet during 13 days, x 768.
Fio. 4. Ductular cell proliferation in liver of duckling fed 0.05% thioacetamide in the diet during days 13 days, X 768.
1371
BILE DUCT HYPERPLASIA TABLE 2.—Dosing experiments
Diet
Vehicle
Dose (mg/0.2 mi.)
4-Dimethylaminoazobenzene
1
1, 2-Dihydroxypropane
0 7.2
3'-Methyl-4-dimethylaminoazobenzene
3
1,2-Dihydroxypropane
Substance
2
Dimethylnitrosamine
Lithocholic acid
Chenodeoxycholic acid
3
3
3
1,2-Dihydroxypropane
Water
1,2-Dihydroxypropane
1,2-Dihydroxypropane
ed from the cycad nut. The other substances tested by Butler (1964) namely carbon tetrachloride and the alkaloids, retrorsine and indicine, did not induce biliary proliferation. A single dose of ethionine or thioacetamide also failed to produce biliary proliferation. The present experiments, however, show that feeding 0.025% ethionine during 14 days, and 0.05% thioacetamide during 13
No. of ducklings
Days
Mortality
Bile duct hyperplasia
5 5
10 10
0 0
0 0
0
2 2
7 14
0 0
0 0
8
2 2
7 14
0 0
0 0
16
2 2
7 14
0 0
0 0
0
2 2
7 14
0 0
0 0
32
2 2
17 14
0 0
0 0
0.04 0.4
5 S
21 21
0 0
0
2
3 3 3
7 14 21
0 0 3
2+ 3+ 4+
4 8
9 9
7 7
5 9
3+ 3+
10
2 2
7 14
0 0
0 0
20
2 2
7 14
0 0
40
2 2
7 14
0 0
+ + + +
10
2 2
7 14
0 0
0 0
20
2 2
7 14
0 0
0 0
40
2 2
7 14
0 0
0 0
+
days induces a clearly denned biliary reaction. The pathology of dimethylnitrosamine poisoning in Pekin ducklings has been studied by Carlton et al. (1966). These authors stated that ducklings are more susceptible to the toxic effects of the drug than rats. Other known carcinogens, namely 1,2-3,4-diepoxybutane, l-oxo-4-nitroquinoline, (3-propionolactone and 4-dimethylami-
1372
R. 0 . VLES
FIG. 5. Bile duct and ductular cell proliferation in liver of duckling fed 0.4% lithocholic acid in the diet during 5 days, X 768.
noazobenzene have been fed to Pekin ducklings by the same authors. These drugs produced none or very few lesions and it was concluded that the duckling has no great advantage over other species as far as screening of potential carcinogens is concerned. Lithocholic acid produces bile duct proliferation in chickens, reptiles, rats, rabbits, mice, guinea pigs, hamsters and monkeys (Eyssen et al., 1965; Leveille and Fairchild, 1965; Carey and Williams, 1965). It is worth remembering here that the socalled "primary" bile acids (cholic and chenodeoxycholic acid) are formed directly from cholesterol in the liver (Anonymous, 1964). As shown in Fig. 7, the "secondary" bile acids (deoxycholic and lithocho-
lie acid) are formed from the "primary" acids by removal of the 7-hydroxy groups by intestinal bacteria. The duckling appears to be an experimental animal which is rather susceptible to the effects of lithocholic acid. Chenodeoxycholic acid produces no ductular cell reaction in ducklings, a result similar to that found in chickens by Leveille and Fairchild (1965). Popper and Schaffner (1961) suggested that the ductular cell reaction could be the result of excretion of an irritating material into the ductules where this material serves as a stimulant for ductular proliferation, inflammatory cell reaction and periductular fibrosis. Lithocholic acid may represent or induce the synthesis of such material. It is conceivable that the same mechanism is involved in the bile duct proliferations observed after administration of aflatoxin, DL-ethionine, thioacetamide or dimethylnitrosamine. SUMMARY
The susceptibility of ducklings to known hepatocarcinogenic substances in producing bile duct proliferation was investigated. Liver changes designated as "biliary ductular cell reaction" and defined as a proliferation of bile ductules and oval cells, collagen BILE
METABOLITES
ACIDS
intestine
liver
'OH CHOLIC ACID
DEOXYCHOLIC
ACID
UTHOCHCKIC
ACID
*
:• . : # . - • >'V;-FIG. 6. Bile duct proliferation in liver of duckling intubated daily with 4 mg. dimethylnitrosamine during 1 week, X 480.
CHENOOEOXYCHOIIC
ACID
FIG. 7. Removal of the 7-hydroxy groups from bile acids by intestinal bacteria.
BILE DUCT HYPERPLASIA
formation and leucocytic infiltration, have been induced in a short time, in ducklings, by DL-ethionine, thioacetamide, dimethylnitrosamine and lithocholic acid. The pathological significance of the ductular cell reaction as well as the possible role of some bile acids in its genesis are discussed. REFERENCES Anonymous, 1964. Lithocholic acid and biliary ductular cellular reaction in animals. Nutr. Rev. 22: 305-306. Ashley, L. M., J. E. Halver, W. K. Gardner and G. N. Wogan, 1965. Crystalline anatoxins cause trout hepatoma. Federation Proc. 24: 627. Asplin, F. D., and R. B. A. Carnaghan, 1961. The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Record, 73: 1215-1218. Butler, W. H., 1964. Acute liver injury in ducklings as a result of anatoxin poisoning. J. Pathol. Bacteriol. 88: 189-195. Butler, W. H., and J. M. Barnes, 1963. Toxic effects of groundnut meal containing aflatoxin to rats and guinea-pigs. Brit. J. Cancer, 17: 699-710. Butler, W. H., and J. M. Barnes, 1966. Carcinoma of the glandular stomach in rats given diets containing anatoxin. Nature, 209: 90. Carey, Jr., J. B., and G. Williams, 1965. Lithocholic acid in human blood serum. Science, 150: 620-622. Carlton, W. W., J. E. Lord and L. Friedman, 1966. Pathology of dimethylnitrosamine-poisoning in Pekin ducklings. Toxicol. Appl. Pharmacol. 8: 224-234. Carnaghan, R. B. A., 1965. Hepatic tumours in ducks fed a low level of toxic groundnut meal. Nature, 208: 308. Chang, S. B., M. M. Abdel Kader, E. L. Wick and G. N. Wogan, 1963. Anatoxin B 2 : chem-
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ical identity and biological activity. Science, 142:1191-1192. Dickens, F., and H. E. H. Jones, 1963. The carcinogenic action of aflatoxin after its subcutaneous injection in the rat. Brit. J. Cancer, 17 : 691-698. Eyssen, H., M. Vandeputte and E. Evrard, 1965. Effect of various dietary bile acids on nutrient absorption and on liver size in chicks. Arch. Inter. Pharmacodyn. 158: 292-306. Hunt, R. D., G. A. Leveille and H. E. Sauberlich, 1963. Dietary bile acids and lipid metabolism II. The ductular cell reaction induced by lithocholic acid. Proc. Soc. Exptl. Biol. Med. 113: 139-142. Lancaster, M. C , F. P. Jenkins and J. McL. Philp, 1961. Toxicity associated with certain samples of groundnuts. Nature, 192: 10951096. Le Breton, E., C. Frayssinet and J. Boy, 1962. Sur l'apparition d'hepatomes "spontanes" chez le rat Wistar. Role de la toxine de ['Aspergillus flavus. Interet en pathologic humaine et cancerologie experimentale. Compt. Rend. 255: 784-786. Leveille, G. A., and D. G. Fairchild, 1965. Lack of effect of dietary chenodeoxycholic acid on plasma and liver lipids and the ductular cell reaction in the chick. Poultry Sci. 44: 16111612. Newberne, P. M., W. W. Carlton and G. N. Wogan, 1964a. Hepatomas in rats and hepatorenal injury in ducklings fed peanut meal or Aspergillus flavus extract. Pathol. Vet. 1: 105-132. Newberne, P. M., G. N. Wogan, W. W. Carlton and M. M. Abdel Kader, 1964b. Histopathologic lesion in ducklings caused by Aspergillus flavus cultures, culture extracts and crystalline anatoxins. Toxicol. Appl. Pharmacol. 6: 542556. Popper, H., and F. Schaffner, 1961. Response of the Liver to Injury in Progress in Liver Diseases. Grune & Stratton, Inc., New York, p. 86. Wogan, G. N., 1965. Editor, Mycotoxins in Foodstuffs. The Massachusetts Institute of Technology Press.
NEWS AND NOTES (Continued from page 1367) from a scientific and practical point of view. An additional highlight will be the symposium on the "Exchange of knowledge in poultry produc-
tion between Europe and the developing countries." In this connection there will be an exhibition displaying the achievements of the European
(Continued on page 1397)