Mucosal Damage Induced by Various Gastric Carcinogens in the Glandular Stomach of the Rat 1,2 Yoshiki Tabuchi,3,
4
Takao Mitsuno,3
and Taketoshi Sugiyama 4,5
Sugimura et al. (1-3) experimentally induced gastric cancer in the rat by continuous oral administrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Aldrich Chemical Co., Milwaukee, Wis.) and provided a useful tool for the investigation of the histogenesis of gastric cancer. We (4) showed that pulse intragastric doses of MNNG acutely injure the glandular mucosa of the rat stomach and that repetition of erosion and repair favors gastric carcinogenesis. This is comparable to an enhanced carcinogenesis in the regenerating target organs already shown in the skin (5J 6), liver (7-10), and bone marrow (11-13), though little is known about the relationship of the above cytocidal effects of carcinogens to cancer production. In the present report, histologic, histochemical, and ultrastructural studies were performed on the process of MNNG-induced erosion, and the process was compared with that with other gastric carcinogens (14-21), erosion-forming chemicals (22-26), and carcinogens for other target organs such as the liver and bone marrow (7J 8J 11 J 13J 27-34)J MATERIALS AND METHODS
Male and female W rats, 6-10 weeks old, were used. They were fed NMF (Oriental Ferment Co., Tokyo, Japan) and given water ad libitum, unless otherwise stated. We studied macroscopically, histologically, and histochemically the acute effects of five known gastric carcinogens: MNNG (1-4), methylnitrosocyanamide (MNC) (14), methylnitrosourea (MNU) (15 J 16), 4-nitroquinoline-I-oxide (NQO) (17J 18), and N-2-fluorenylacetamide (2-FAA) (19-21); the gastric erosion-forming compound with alkylating capacity, iodoacetamide
(lAM) (22-24); the same without mutagenicity, acetylsalicylic acid (aspirin) (25 J 26); carcinogens for other target organs such as dimethylnitrosamine (DMN) (8J 27), 3'methyl-4-dimethylaminoazobenzene (3'-Me-DAB) (28J 29), ,e-naphthylamine (30), N-nitroso-N-butylurea (NBU) (31 J 32), DL-ethionine (33 J 34), and 7,I2-dimethylbenz[a]anthracene (DMBA) (7 J 11 J 13); and boiling water as a physical factor. 6 For MNNG, the process of erosion formation was pursued also ultrastructurally. For MNNG, aspirin, and lAM.. and each of other chemicals, 45, 12, and 42 animals were used, respectively. Three control animals were used for each chemical. The typical procedure for the induction of erosion was intermittent, heavy intragastric doses of the chemicals for 3 days. The animals were deprived of food for 24 hours and given a single intragastric treatment. The second and third doses were given 24 and 48 hours after the first treatment without restriction of food intake. Because of the different solubility of these compounds, MNNG, lAM, and DMN were dissolved in distilled water; MNC, MNU, NQO, and NBU were dissolved in 25% ethanol. DMBA, 2-FAA, p-naphthylamine, and 3Me-DAB were suspended in water with a Teflon pestle or dissolved in sesame oil. Aspirin was homogenized in 0.1 N HCl solution as described (25 J 26). The chemicals dissolved in 2 ml distilled water and 25% ethanol were given to the animals through a stomach tube. Various dose levels (0-2.00 mM jkg body wt) were tested for the detection of the optimal doses for erosion formation after 3 daily doses. Six animals were subjected to 20% full-thickness scald burns with the use of boiling water at 99° C for 10 seconds (35). / Usually 12 hours after the second and third treatments, 3 animals were killed under ether anesthesia. For MNNG-induced erosion, 5 treated rats were also killed 0.5, I, 2, 4, 8, and 12 hours after the first MNNG treatment. Mucosal changes were investigated 12 and 24 hours after the thermal burn. At autopsy, the duodenum was ligated and 4 mI of 10% neutral formalin was injected into the gastric lumen. The stomach was removed, fixed in 10% neutral formalin, and studied macroscopically and microscopically. Microscopic sections were stained with hematoxylin and eosin, Sudan Received May 6, 1975; accepted July 31, 1975. Supported in part by grants 801059, 801070, and 801550 from the Ministry of Education. 3 Department of Surgery, Kobe University School of Medicine, Ikuta-ku, Kobe 650, Japan. 4 Department of Pathology, Kobe University School of Medicine. 5 We are indebted to Professor Hideya Endo, Director of the Cancer Institute, Kyushu University, Fukuoka 812, Japan, for providing methylnitrosocyanamide and methylnitrosourea. 6 The following is a list of the products used and the suppliers. NQO and lAM: Nakarai Chemical Ltd., Kyoto, Japan; 2-FAA and DL-ethionine: Wako Pure Chemical Co., Osaka, Japan; aspirin: Bayer Co., Leverkusen, West Germany; DMN: Tokyo Chemical Synthetic Industry Co., Tokyo; NBU: Izumi Chemical Research Lab., Tokyo; DMBA: Eastman Organic Chemicals, Rochester, N.Y. MNC and MNU were synthesized and supplied by Professor H. Endo; 3'-Me-DAB and ,e-naphthylamine were gifts from Dr. N. Odashima, National Institute of Hygienic Sciences, Tokyo. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.6, DECEMBER 1975
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SUMMARY-The process of erosion formation in the glandular stomach of the rat given single and multiple intragastric doses of 100 mg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)/kg body weight, was studied histologically, histochemically, and ultramicroscopically and compared with erosion induced by other gastric carcinogens and erosion-forming chemicals. The acute effect of several nongastric carcinogens on the glandular mucosa was also studied. The earliest degenerative transformation, fatty change, was found in the surface mucous cells within 1 hour after a one-pulse intragastric dose of 100 mg MNNG/kg body weight; the change gradually progressed into deeper glandular cells and after three successive doses, erosion was complete in every rat. Ultrastructurally, four main glandular cells showed essentially similar dege'nerative alterations. Fatty change was also induced by other gastric carcinogens such as 4-nitroquinoline-l-oxide, .. methylnitrosocyanamide, methylnitrosourea, N-2-f1uorenylacetamide, and iodoacetamide, a noncarcinogenic alkylating agent. Mucosal damage induced by acetylsalicylic acid and thermal burn did not .show fatty change. Nongastric carcinogens failed to induce mucosar damage. The relationship of the carcinogen-induced fatty change and mucosal damage to carcinogenesis was discussed.-J Natl Cancer Inst 55: 1395-1401, 1975.
1396
TABUCHI, MITSUNO, AND SUGIYAMA
III, toluidine blue, alcian blue, and periodic acid-Schiff (PAS). For electron microscopy, the stomach was cut along the greater curvature and inspected for mucosal changes; the lesions, if any, were fixed in 1% osmium tetroxide buffered with 0.1 M cacodylic acid sodium salt (pH 7.3-7.4), dehydrated in a series of ethanol, dipped in propylene oxide, a~d embedded in Epon 812. For .observation of the earlIest changes, several mucosal SItes were studied. The ultrathin sections were stained by uranyl acetate and lead citrate and examined under a Hitachi HU-IID electron microscope. RESULTS Mucosal Damage Induced by MNNG
Histochemical Changes After MNNG Treatment
The PAS-positive mucous granules were reduced in the surface mucous cells within I hour in the above predirected sites and later also in the other regions. The granules in the mucous neck cells were reduced 8-12 hours after MNNG treatment. Another prominent histochemical transformation was fatty change of the mucosal cells. This first appeared in the surface mucous cells within 1 hour and became manifest within 2 hours (figs. TABLE
Ultramicroscopic Changes After MNNG Treatment
Electron microscopically, all four types of mucosal cells-surface mucous, mucous neck, and parietal and chief cells-revealed essentially similar degenerative changes after MNNG treatment, though these progressed from the surface to the deeper mucosal cells according to their distribution. The earliest ultramicroscopic variations were a reduction of secretory granules, an increase of enlarged lysosomes, and the appearance of autophagic vacuoles containing debris of cell organelles such as secretory granules, mitochondria, and various membranous structures (figs. 4A, B). These transformations were observed in the surface mucous cells as early as I hour and later in the deeper cells. Some autophagic vacuoles contained electron-opaque bodies, often with a vacuole in the center, possibly fat droplets (figs. 4A, B). However, some dilated cisternae of the rough endoplasmic reticulum contained similar electron-opaque fat droplets. Many larger fat droplets around the endoplasmic reticulum appeared to be derived from the smaller ones by fusion outside the cisternae of the endoplasmic reticulum. They were close to the membranes of the endoplasmic reticulum (fig. 5). These results seemed to indicate that fat droplets originated in the cisternae of the endoplasmic reticulum. Many moderately damaged cells revealed a few or numerous electron-opaque fat droplets. They were irregularly but sharply outlined, located in groups, or scattered diffusely in the cytoplasm (figs. 6, 7). In these cells, secretory granules were absent and the cytoplasmic matrix was decreased. Mitochondria were reduced in number and size and showed an increased density. Golgi lamellae and vesicles were also fewer; rough endoplasmic reticulum was vesiculated and degranulated. Concentric lamellar bodies composed of degenerated smooth endoplasmic reticulum were sometimes observed in the moderately damaged parietal cells. Autophagic
I.-Mucosal erosion in glandular stomach of the rat induced by various chemicals after three-pulse intragastric doses of chemicals
Range of dose treated Molecular weight
Chemicals
mg/kg body wt (mM/kg body wt)
Mucosal change
Optimal dose a (mM/kg body wt)
Erosion
Fatty change
Gastric carcinogens: MNNG - - - - - - - - - - - - - - - - - - -NQO - _- _- _- - _- - - -MNC _ MNlJ _ 2-FAA - _
147 190 85 103 223
0-100 0- 14 0- 50 0-140 0-200
(0.680) (0.074) (0.588) (1.346) (0.896)
0.680 0.037 0.294 0.673 0.448
+ + + + +
+ + + + +
Erosion-forming chemicals: IAM Aspirin -------
_ _
184 180
0- 60 (0.326) 0-128 (1.226)
0.163 0.633
+ +
+
N ongastric DMN carcinogen:
_
74 293 143 163 256 145
0-150 0-480 0-290 0-320 0-510 0-290
3'-Me-DAB Ii-Naphthylamine Direthionine
DMBA NBD Burn
- - _- ---_ _
- - - - - - __ - - _- - - ----- - - __ - __ _
.. Optimal single dose for gastric erosion after three-pulse doses. ~
See (36).
(2.000) (2.000) (2.000) (2.000) (2.000) (2.000)
O'Neill's method b
+
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Superficial mucosal damage was induced in every rat 12 hours after a single dose of 100 mg MNNGjkg body weight. After three doses, every rat revealed erosion with destruction of the whole mucosal layer. Erosion was preferentially induced in the fundal mucosa along the transverse ridge and in the border between fundal and antral regions. They were considered the predirected sites for erosion formation. Erosion was usually covered with buffy material composed of cell debris and surrounded by a slightly hyperemic zone (fig. 1). Histologically, surface mucous cells lining the gastric lumen were vacuolated within 1 hour, deeper mucosal cells gradually degenerated, and circulatory disturbance such as congestion and hemorrhage was observed in the mucosa within several hours. Complete erosion was formed after three successive doses. Similar observations were already made in (4).
2A, B). The cells in the deeper mucosa, submucosa, and other regions also contained fat droplets on days 2 and 3, though the submucosal change was less prominent when compared with the mucosal alteration (fig. 3).
q-IEMICALL Y INDUCED GASTRIC MUCOSAL DAMAGE
vacuoles were almost lost in this stage. Nuclear chromatin was clumped and disorganized, and nucleoli were reduced in size and number. Definite nucleolar segregation was not observed. In general, cytoplasmic changes were more prominent than those of the nucleus. As the changes became severe with time, large collection of fat droplets, loss of cytoplasmic organelles, nuclear karyolysis, and finally destruction of cell membrane and junctional complex killed the cells. Mucosal Damage Induced by Other Chemicals
DISCUSSION
The MNNG-induced mucosal damage in the glandular stomach started in the surface epithelium within I hour, and erosion was formed within 2-3 days after three successive intragastric doses of 100 mg MNNG/kg body weight. The histologic studies performed in parallel with ultramicroscopic and histochemical studies revealed that MNNG action on the mucosal cells was direct; the mucosal cells were damaged from the surface to the bottom according to their distribution, and no selective tendency of damaging special kinds of mucosal cells was found. The present studies also confirmed that other gastric carcinogens such as NQO, MNC, MNU, and 2-FAA induced erosion in the same manner as MNNG. The alkylating agent (22) lAM, when administered orally for as few as 175 days, is regarded noncarcinogenic to the stomach and also induces erosion. However, nongastric carcinogens such as DMN, 3'-Me-DAB, f3-naphthylamine, NBU, DMBA, and DL-ethionine failed to induce mucosal damage. The histologic, histochemical, and ultramicroscopic studies revealed that the earliest transformation was fatty change in the mucosal cells after MNNG treatments. This alteration was also induced by other erosion-forming chemicals such as NQO, MNC, MNU, 2FAA, and lAM. Ultrastructurally, the earliest change was the formation of small electron-opaque fat droplets within the cisternae of the endoplasmic reticulum. They coalesced to form larger droplets with advanced cell damage. The fat droplets seemed to contain triglyceride or saturated fat, judged from their low electron density.
It is. unknown whether the fat droplets within autop.hagic vacuoles were derived from the ingested materIals or the fat droplets taken from the outside. However, they may have a less important function in the fatty change (36) 37) of the mucosal cells, since the occurrence of the fat-containing autophagic vacuoles was n.ot frequent. T~us conceivably the fat droplets are derIved from the CIsternae of the rough endoplasmic reticulum. . It has been i.nd~cated from the studies of fatty liver mduced by ethIOnine {38} 39), CCl4 (38-41), orotic acid (38}.42), and choline deficiency (38) 39} 43) that the earlIest fat droplets appear in the dilated cisternae of the endoplasmic reticulum and constitute "liposomes" (38) 39). T~e defec~ in the removal of the fat droplets from the CIsternae Induced by these chemicals is caused by inhibited synthesis of proteins, phospholipids, and cholesterols that are required for assembling the secretory form~ of lipids or lipoproteins (39) 40} 43). Therefore, possIbly the fatty change of the mucosal cells induced ?y various gastric carcinogens is essentially the same wIth the fatty change of the liver induced by various hepatic carcinogens such as DMN (8) 27), 3'-MeI?AB (28) 29),. and ethionine (28) 38} 39). Since puromyCIn (44) also Induces fatty change of the liver, and the comp~>unds damaging th~ gastric mucosa in the present experImen~s .w~re ca~cInogens or. alkylating agents known to InhIbIt proteIn and nucleIc acid synthesis, the fatty ch~ng~ ?f the mucosal cells may be an expression of such InhIbItory effects on cell metabolism. Such an in~ibitory effect on cells may have an important function In cell death. Although cytolysis does not lead to cancer, many carcinogens damage their specific target cells cytogenetically and morphologically as an early effect; the effects parallel with carcinogenicity for many chemicals (7) 9-J3} 18} 28). Therefore, it is possible that the target DNA for the chemicals in cytolysis and carcinogenesis is essentially th~ same; thus both processes are correlated. ~urth~r studIes are nece~sary to solve the positive rela~IOnship .between cytolysIs and carcinogenesis with varIOUS carcInogens. REFERENCES
(I) SUGIMURA T, FUJIMURA S: Tumour production in glandular stomach of rat by N-methyl-N'-nitro-N-nitrosoguanidine. Nature 216:943-944, 1967 (2) SUGIMURA T, FUJIMURA S, BABA T: Tumor production in the glandular stomach and alimentary tract of the rat by N-methyl-N'-nitro-N-nitrosoguanidine. Cancer Res 30:455465, 1970 (J) SUGIMURA T, KAWACHI T: Experimental stomach cancer. In Methods in Cancer Research (Busch H, ed.), vol 7. New York and London, Academic Press, 1973, pp 245-307 (4) TABUCHI Y, OGINO T, MITSUNO T, et al: Possible role of mucosal damage in stomach carcinogenesis with N-methyl-N'nitro-N-nitrosoguanidine. J Natl Cancer Inst 52: 1589-1594, 1974 (5) YAMAGIWA K, IcHIKAWA K: Artificial production of cancers. Gann 10:249-290, 1915 (6) POUND AW, WITHERS HR: The influence of some irritant chemicals and scarification on tumour production by urethane in mice. Br J Cancer 17:460-470, 1963 (7) MARQUARDT H, STERNBERG SS, PHILIPS FS: 7,12-dimethylbenz(a)anthracene and hepatic neoplasia in regenerating rat liver. Chern BioI Interact 2:401-403, 1970 (8) MAGEE PN, BARNES JM: The production of malignant hepatic tumours in the rat by feeding dimethylnitrosamine. Br J Cancer 10: 114-122, 1956 (9) WOGAN. GN,. ED~ARDS .~S, NEwBERN~ PM:. ~tructure-activity relatIOnshIps III tOXICIty and carcInogenICIty of aflatoxins and analogs. Cancer Res 31: 1936-1942, 1971 (10) MIRONESCU S, ClOviRNACHE M: Mitotic, chromosomal, and nucleolar alternations induced by thioacetamide in relation
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The extent of the gastric lesion was judged from the width and depth of erosion. NQO, MNC, MNU, and lAM induced similar erosion in the predirected regions for MNNG erosion in every rat (figs. 8-10). The amount of these chemicals necessary for erosion after three successive doses is shown in table 1. MNC was 2.3 times more potent in inducing damage than MNNG; NQO was the strongest on the same molecular-weight basis. The erosion induced by 2-FAA, aspirin, and thermal burn was not localized in the specific mucosal regions but scattered over the wide areas of fundal mucosa. Histologically, these chemicals destroyed the whole layer of the glandular mucosa. The mucosal cells around the erosion were vacuolated and revealed a reduced amount of PAS-positive granules; the mucosa was congested and hemorrhagic. Fatty change was also induced in the mucosal cells after treatment with NQO, MNC, MNU, 2FAA, and lAM (figs. 8-10), whereas aspirin and thermal burn did not induce fatty change. Nongastric carcinogens such as DMN, 3'-Me-DAB, f3naphthylamine, NBU, DL-ethionine, and DMBA failed to . induce gastric mucosal damage,. even with a large dose of 2.0 roM/kg body weight and with a longer observation time up to 10 days after the last dose (table I).
1397
1398
(11) (12) (13)
(14)
(15)
(16)
(18) (19)
(20) (21)
(22)
(23) (24)
(25) (26) (27)
to the mitotic cycle after partial hepatectomy. J Natl Cancer Inst 46:49-61, 1971 HUGGINS C, SUGIYAMA T: Induction of leukemia in rat by pulse doses of 7,12-dimethylbenz(a)anthracene. Proc Nat! Acad Sci USA 55:74-81, 1966 HUGGINS C, GRAND L, OKA K: Hundred day leukemia. Preferential induction in rat by pulse-doses of 7,8,12-trimethylbenz(a)anthracene. J Exp Med 131:321-330, 1970 SUGIYAMA T: Role of erythroprotein in 7,12-dimethylbenz(a)anthracene in induction of acute chromosome aberration and leukemia in the rat. Proc Natl Acad Sci USA 68:27612764, 1971 ENDO H, TAKAHASHI K, KINOSHITA N, et al: Production of gastric and esophageal tumors in rats by methylnitrosocyanamide, a possible candidate of etiologic factors for human gastric cancer. Proc Jpn Acad. In press DRUCKREY H, IVANKOVIC S, BUCHELER J, et al: Erzeugung von Magen- und Pankreas-Krebs beim Meerschweinchen durch Methylnitroso-harnstoff und -urethan. Z Krebsforsch 71: 167182, 1968 MIRVISH SS: Kinetics of dimethylamine nitrosation in relation to nitrosamine carcinogenesis. J Nat! Cancer Inst 46: 11831193, 1971 BABA T, MISU Y, TAKAYAMA S: Induction of cancer of the glandular stomach in a rat. A new form of experiment. Gann 53:381-387, 1962 MORI K: Experimental gastric cancer with 4-nitroquinoline I-oxide and its derivatives. Gann Monogr 8:233-261, 1969 MORRIS HP, WAGNER BP, RAY FE, et al: Comparative study of cancer and other lesions of rats fed N,N'-2,7-fluorenylenebisacetamide or N-2-fiuorenylacetamide. Natl Cancer Inst Monogr 5:1-30, 1961 STEWART HL, SNELL KC, MORRIS HP, et al: Carcinoma of the glandular stomach of rats ingesting N,N'-2,7-fluorenylenebisacetamide. Nat! Cancer Inst Monogr 5: 105-139, 1961 NAGAYO T: Effect of oral administration of N,N'-2,7-fluorenylenebisacetamide combined with traumatic ulcer of the glandular stomach of Buffalo rats. J Nat! Cancer Inst 35:829-840, 1965 FRUCHTER RG, CRESTFIED AM: The specific alkylation by iodoacetamide of histidine-12 in the active site of ribonuclease. J BioI Chern 22:5807-5812, 1967 LAUCH 11: lodoacetamide induced gastric ulcer in rats. Proc Soc Exp BioI Med 109:905-908, 1962 YASHIN R, LEESE CL: The production of chronic gastritis and ulceration in the glandular stomach of rats by iodoacetamide (lAM). Eur J Cancer 6:425-432, 1970 HINGSON DJ, ITO S: Effect of aspirin and related compounds on the fine structure of mouse gastric mucosa. Gastroenterology 61:156-177,1971 LEB R, SIEGEL HI, GLASS GB: Effects of salicylates on the canine stomach: A morphological and histochemical study. Gastroenterology 62:970-979, 1972 SCHMALL D, PREUs..
4
Takao Mitsuno,3
and Taketoshi Sugiyama 4,5
Sugimura et al. (1-3) experimentally induced gastric cancer in the rat by continuous oral administrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Aldrich Chemical Co., Milwaukee, Wis.) and provided a useful tool for the investigation of the histogenesis of gastric cancer. We (4) showed that pulse intragastric doses of MNNG acutely injure the glandular mucosa of the rat stomach and that repetition of erosion and repair favors gastric carcinogenesis. This is comparable to an enhanced carcinogenesis in the regenerating target organs already shown in the skin (5J 6), liver (7-10), and bone marrow (11-13), though little is known about the relationship of the above cytocidal effects of carcinogens to cancer production. In the present report, histologic, histochemical, and ultrastructural studies were performed on the process of MNNG-induced erosion, and the process was compared with that with other gastric carcinogens (14-21), erosion-forming chemicals (22-26), and carcinogens for other target organs such as the liver and bone marrow (7J 8J 11 J 13J 27-34)J MATERIALS AND METHODS
Male and female W rats, 6-10 weeks old, were used. They were fed NMF (Oriental Ferment Co., Tokyo, Japan) and given water ad libitum, unless otherwise stated. We studied macroscopically, histologically, and histochemically the acute effects of five known gastric carcinogens: MNNG (1-4), methylnitrosocyanamide (MNC) (14), methylnitrosourea (MNU) (15 J 16), 4-nitroquinoline-I-oxide (NQO) (17J 18), and N-2-fluorenylacetamide (2-FAA) (19-21); the gastric erosion-forming compound with alkylating capacity, iodoacetamide
(lAM) (22-24); the same without mutagenicity, acetylsalicylic acid (aspirin) (25 J 26); carcinogens for other target organs such as dimethylnitrosamine (DMN) (8J 27), 3'methyl-4-dimethylaminoazobenzene (3'-Me-DAB) (28J 29), ,e-naphthylamine (30), N-nitroso-N-butylurea (NBU) (31 J 32), DL-ethionine (33 J 34), and 7,I2-dimethylbenz[a]anthracene (DMBA) (7 J 11 J 13); and boiling water as a physical factor. 6 For MNNG, the process of erosion formation was pursued also ultrastructurally. For MNNG, aspirin, and lAM.. and each of other chemicals, 45, 12, and 42 animals were used, respectively. Three control animals were used for each chemical. The typical procedure for the induction of erosion was intermittent, heavy intragastric doses of the chemicals for 3 days. The animals were deprived of food for 24 hours and given a single intragastric treatment. The second and third doses were given 24 and 48 hours after the first treatment without restriction of food intake. Because of the different solubility of these compounds, MNNG, lAM, and DMN were dissolved in distilled water; MNC, MNU, NQO, and NBU were dissolved in 25% ethanol. DMBA, 2-FAA, p-naphthylamine, and 3Me-DAB were suspended in water with a Teflon pestle or dissolved in sesame oil. Aspirin was homogenized in 0.1 N HCl solution as described (25 J 26). The chemicals dissolved in 2 ml distilled water and 25% ethanol were given to the animals through a stomach tube. Various dose levels (0-2.00 mM jkg body wt) were tested for the detection of the optimal doses for erosion formation after 3 daily doses. Six animals were subjected to 20% full-thickness scald burns with the use of boiling water at 99° C for 10 seconds (35). / Usually 12 hours after the second and third treatments, 3 animals were killed under ether anesthesia. For MNNG-induced erosion, 5 treated rats were also killed 0.5, I, 2, 4, 8, and 12 hours after the first MNNG treatment. Mucosal changes were investigated 12 and 24 hours after the thermal burn. At autopsy, the duodenum was ligated and 4 mI of 10% neutral formalin was injected into the gastric lumen. The stomach was removed, fixed in 10% neutral formalin, and studied macroscopically and microscopically. Microscopic sections were stained with hematoxylin and eosin, Sudan Received May 6, 1975; accepted July 31, 1975. Supported in part by grants 801059, 801070, and 801550 from the Ministry of Education. 3 Department of Surgery, Kobe University School of Medicine, Ikuta-ku, Kobe 650, Japan. 4 Department of Pathology, Kobe University School of Medicine. 5 We are indebted to Professor Hideya Endo, Director of the Cancer Institute, Kyushu University, Fukuoka 812, Japan, for providing methylnitrosocyanamide and methylnitrosourea. 6 The following is a list of the products used and the suppliers. NQO and lAM: Nakarai Chemical Ltd., Kyoto, Japan; 2-FAA and DL-ethionine: Wako Pure Chemical Co., Osaka, Japan; aspirin: Bayer Co., Leverkusen, West Germany; DMN: Tokyo Chemical Synthetic Industry Co., Tokyo; NBU: Izumi Chemical Research Lab., Tokyo; DMBA: Eastman Organic Chemicals, Rochester, N.Y. MNC and MNU were synthesized and supplied by Professor H. Endo; 3'-Me-DAB and ,e-naphthylamine were gifts from Dr. N. Odashima, National Institute of Hygienic Sciences, Tokyo. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.6, DECEMBER 1975
1395
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SUMMARY-The process of erosion formation in the glandular stomach of the rat given single and multiple intragastric doses of 100 mg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)/kg body weight, was studied histologically, histochemically, and ultramicroscopically and compared with erosion induced by other gastric carcinogens and erosion-forming chemicals. The acute effect of several nongastric carcinogens on the glandular mucosa was also studied. The earliest degenerative transformation, fatty change, was found in the surface mucous cells within 1 hour after a one-pulse intragastric dose of 100 mg MNNG/kg body weight; the change gradually progressed into deeper glandular cells and after three successive doses, erosion was complete in every rat. Ultrastructurally, four main glandular cells showed essentially similar dege'nerative alterations. Fatty change was also induced by other gastric carcinogens such as 4-nitroquinoline-l-oxide, .. methylnitrosocyanamide, methylnitrosourea, N-2-f1uorenylacetamide, and iodoacetamide, a noncarcinogenic alkylating agent. Mucosal damage induced by acetylsalicylic acid and thermal burn did not .show fatty change. Nongastric carcinogens failed to induce mucosar damage. The relationship of the carcinogen-induced fatty change and mucosal damage to carcinogenesis was discussed.-J Natl Cancer Inst 55: 1395-1401, 1975.
1396
TABUCHI, MITSUNO, AND SUGIYAMA
III, toluidine blue, alcian blue, and periodic acid-Schiff (PAS). For electron microscopy, the stomach was cut along the greater curvature and inspected for mucosal changes; the lesions, if any, were fixed in 1% osmium tetroxide buffered with 0.1 M cacodylic acid sodium salt (pH 7.3-7.4), dehydrated in a series of ethanol, dipped in propylene oxide, a~d embedded in Epon 812. For .observation of the earlIest changes, several mucosal SItes were studied. The ultrathin sections were stained by uranyl acetate and lead citrate and examined under a Hitachi HU-IID electron microscope. RESULTS Mucosal Damage Induced by MNNG
Histochemical Changes After MNNG Treatment
The PAS-positive mucous granules were reduced in the surface mucous cells within I hour in the above predirected sites and later also in the other regions. The granules in the mucous neck cells were reduced 8-12 hours after MNNG treatment. Another prominent histochemical transformation was fatty change of the mucosal cells. This first appeared in the surface mucous cells within 1 hour and became manifest within 2 hours (figs. TABLE
Ultramicroscopic Changes After MNNG Treatment
Electron microscopically, all four types of mucosal cells-surface mucous, mucous neck, and parietal and chief cells-revealed essentially similar degenerative changes after MNNG treatment, though these progressed from the surface to the deeper mucosal cells according to their distribution. The earliest ultramicroscopic variations were a reduction of secretory granules, an increase of enlarged lysosomes, and the appearance of autophagic vacuoles containing debris of cell organelles such as secretory granules, mitochondria, and various membranous structures (figs. 4A, B). These transformations were observed in the surface mucous cells as early as I hour and later in the deeper cells. Some autophagic vacuoles contained electron-opaque bodies, often with a vacuole in the center, possibly fat droplets (figs. 4A, B). However, some dilated cisternae of the rough endoplasmic reticulum contained similar electron-opaque fat droplets. Many larger fat droplets around the endoplasmic reticulum appeared to be derived from the smaller ones by fusion outside the cisternae of the endoplasmic reticulum. They were close to the membranes of the endoplasmic reticulum (fig. 5). These results seemed to indicate that fat droplets originated in the cisternae of the endoplasmic reticulum. Many moderately damaged cells revealed a few or numerous electron-opaque fat droplets. They were irregularly but sharply outlined, located in groups, or scattered diffusely in the cytoplasm (figs. 6, 7). In these cells, secretory granules were absent and the cytoplasmic matrix was decreased. Mitochondria were reduced in number and size and showed an increased density. Golgi lamellae and vesicles were also fewer; rough endoplasmic reticulum was vesiculated and degranulated. Concentric lamellar bodies composed of degenerated smooth endoplasmic reticulum were sometimes observed in the moderately damaged parietal cells. Autophagic
I.-Mucosal erosion in glandular stomach of the rat induced by various chemicals after three-pulse intragastric doses of chemicals
Range of dose treated Molecular weight
Chemicals
mg/kg body wt (mM/kg body wt)
Mucosal change
Optimal dose a (mM/kg body wt)
Erosion
Fatty change
Gastric carcinogens: MNNG - - - - - - - - - - - - - - - - - - -NQO - _- _- _- - _- - - -MNC _ MNlJ _ 2-FAA - _
147 190 85 103 223
0-100 0- 14 0- 50 0-140 0-200
(0.680) (0.074) (0.588) (1.346) (0.896)
0.680 0.037 0.294 0.673 0.448
+ + + + +
+ + + + +
Erosion-forming chemicals: IAM Aspirin -------
_ _
184 180
0- 60 (0.326) 0-128 (1.226)
0.163 0.633
+ +
+
N ongastric DMN carcinogen:
_
74 293 143 163 256 145
0-150 0-480 0-290 0-320 0-510 0-290
3'-Me-DAB Ii-Naphthylamine Direthionine
DMBA NBD Burn
- - _- ---_ _
- - - - - - __ - - _- - - ----- - - __ - __ _
.. Optimal single dose for gastric erosion after three-pulse doses. ~
See (36).
(2.000) (2.000) (2.000) (2.000) (2.000) (2.000)
O'Neill's method b
+
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Superficial mucosal damage was induced in every rat 12 hours after a single dose of 100 mg MNNGjkg body weight. After three doses, every rat revealed erosion with destruction of the whole mucosal layer. Erosion was preferentially induced in the fundal mucosa along the transverse ridge and in the border between fundal and antral regions. They were considered the predirected sites for erosion formation. Erosion was usually covered with buffy material composed of cell debris and surrounded by a slightly hyperemic zone (fig. 1). Histologically, surface mucous cells lining the gastric lumen were vacuolated within 1 hour, deeper mucosal cells gradually degenerated, and circulatory disturbance such as congestion and hemorrhage was observed in the mucosa within several hours. Complete erosion was formed after three successive doses. Similar observations were already made in (4).
2A, B). The cells in the deeper mucosa, submucosa, and other regions also contained fat droplets on days 2 and 3, though the submucosal change was less prominent when compared with the mucosal alteration (fig. 3).
q-IEMICALL Y INDUCED GASTRIC MUCOSAL DAMAGE
vacuoles were almost lost in this stage. Nuclear chromatin was clumped and disorganized, and nucleoli were reduced in size and number. Definite nucleolar segregation was not observed. In general, cytoplasmic changes were more prominent than those of the nucleus. As the changes became severe with time, large collection of fat droplets, loss of cytoplasmic organelles, nuclear karyolysis, and finally destruction of cell membrane and junctional complex killed the cells. Mucosal Damage Induced by Other Chemicals
DISCUSSION
The MNNG-induced mucosal damage in the glandular stomach started in the surface epithelium within I hour, and erosion was formed within 2-3 days after three successive intragastric doses of 100 mg MNNG/kg body weight. The histologic studies performed in parallel with ultramicroscopic and histochemical studies revealed that MNNG action on the mucosal cells was direct; the mucosal cells were damaged from the surface to the bottom according to their distribution, and no selective tendency of damaging special kinds of mucosal cells was found. The present studies also confirmed that other gastric carcinogens such as NQO, MNC, MNU, and 2-FAA induced erosion in the same manner as MNNG. The alkylating agent (22) lAM, when administered orally for as few as 175 days, is regarded noncarcinogenic to the stomach and also induces erosion. However, nongastric carcinogens such as DMN, 3'-Me-DAB, f3-naphthylamine, NBU, DMBA, and DL-ethionine failed to induce mucosal damage. The histologic, histochemical, and ultramicroscopic studies revealed that the earliest transformation was fatty change in the mucosal cells after MNNG treatments. This alteration was also induced by other erosion-forming chemicals such as NQO, MNC, MNU, 2FAA, and lAM. Ultrastructurally, the earliest change was the formation of small electron-opaque fat droplets within the cisternae of the endoplasmic reticulum. They coalesced to form larger droplets with advanced cell damage. The fat droplets seemed to contain triglyceride or saturated fat, judged from their low electron density.
It is. unknown whether the fat droplets within autop.hagic vacuoles were derived from the ingested materIals or the fat droplets taken from the outside. However, they may have a less important function in the fatty change (36) 37) of the mucosal cells, since the occurrence of the fat-containing autophagic vacuoles was n.ot frequent. T~us conceivably the fat droplets are derIved from the CIsternae of the rough endoplasmic reticulum. . It has been i.nd~cated from the studies of fatty liver mduced by ethIOnine {38} 39), CCl4 (38-41), orotic acid (38}.42), and choline deficiency (38) 39} 43) that the earlIest fat droplets appear in the dilated cisternae of the endoplasmic reticulum and constitute "liposomes" (38) 39). T~e defec~ in the removal of the fat droplets from the CIsternae Induced by these chemicals is caused by inhibited synthesis of proteins, phospholipids, and cholesterols that are required for assembling the secretory form~ of lipids or lipoproteins (39) 40} 43). Therefore, possIbly the fatty change of the mucosal cells induced ?y various gastric carcinogens is essentially the same wIth the fatty change of the liver induced by various hepatic carcinogens such as DMN (8) 27), 3'-MeI?AB (28) 29),. and ethionine (28) 38} 39). Since puromyCIn (44) also Induces fatty change of the liver, and the comp~>unds damaging th~ gastric mucosa in the present experImen~s .w~re ca~cInogens or. alkylating agents known to InhIbIt proteIn and nucleIc acid synthesis, the fatty ch~ng~ ?f the mucosal cells may be an expression of such InhIbItory effects on cell metabolism. Such an in~ibitory effect on cells may have an important function In cell death. Although cytolysis does not lead to cancer, many carcinogens damage their specific target cells cytogenetically and morphologically as an early effect; the effects parallel with carcinogenicity for many chemicals (7) 9-J3} 18} 28). Therefore, it is possible that the target DNA for the chemicals in cytolysis and carcinogenesis is essentially th~ same; thus both processes are correlated. ~urth~r studIes are nece~sary to solve the positive rela~IOnship .between cytolysIs and carcinogenesis with varIOUS carcInogens. REFERENCES
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The extent of the gastric lesion was judged from the width and depth of erosion. NQO, MNC, MNU, and lAM induced similar erosion in the predirected regions for MNNG erosion in every rat (figs. 8-10). The amount of these chemicals necessary for erosion after three successive doses is shown in table 1. MNC was 2.3 times more potent in inducing damage than MNNG; NQO was the strongest on the same molecular-weight basis. The erosion induced by 2-FAA, aspirin, and thermal burn was not localized in the specific mucosal regions but scattered over the wide areas of fundal mucosa. Histologically, these chemicals destroyed the whole layer of the glandular mucosa. The mucosal cells around the erosion were vacuolated and revealed a reduced amount of PAS-positive granules; the mucosa was congested and hemorrhagic. Fatty change was also induced in the mucosal cells after treatment with NQO, MNC, MNU, 2FAA, and lAM (figs. 8-10), whereas aspirin and thermal burn did not induce fatty change. Nongastric carcinogens such as DMN, 3'-Me-DAB, f3naphthylamine, NBU, DL-ethionine, and DMBA failed to . induce gastric mucosal damage,. even with a large dose of 2.0 roM/kg body weight and with a longer observation time up to 10 days after the last dose (table I).
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