aroclor1254-inducedintestinalmetaplasiaandadenocarcinomainthe...

(CANCER RESEARCH 41, 5052-5059, December 1981]0008-5472/81 /0041-OOOOS02.00

Aroclor 1254-induced intestinal Metaplasia and Adenocarcinoma in theGlandular Stomach of F344 Rats1

Robin W. Morgan,2 Jerrold M. Ward, and Philip E. Hartman3

Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 [P. W. M., P. E. H.J, and Tumor Pathology and Pathogenesis Section,Laboratory of Comparative Carcinogenesis, Division of Cancer Cause and Prevention. National Cancer Institute, Frederick Cancer Research Center NIHFrederick, Maryland 21701 [J. M. W.J

ABSTRACT

Ingestion of diets containing Aroclor 1254, a mixture ofpolychlorinated biphenyls, for 2 years led to a dose-related

increase in the incidence of focal lesions in the glandularstomachs of male and female F344 rats. The incidence ofstomach lesions was 6% in control specimens and in specimens from rats fed a diet containing 25, 50, or 100 ppmAroclor 1254, the incidences of stomach lesions were 10, 17,and 35%, respectively. The majority of gastric lesions in treatedrats were histologically identified as intestinal metaplasia characterized by an architecture resembling that of intestinal cryptsand particularly by goblet cells, which stained with Alcian blueand periodic acid-Schiff reagent. Adenocarcinomas were found

in six specimens. Most (88%) of the lesions were located in thepyloric region of the glandular stomach. No multiple lesionswere observed among 47 control specimens examined; however, nine cases of multiple lesions were observed in 30 lesion-containing specimens from Aroclor 1254-treated rats. Althoughthe exact relationship between gastric intestinal metaplasia andadenocarcinoma remains to be established, they commonlycoexist and may share initiating mechanisms.

INTRODUCTION

RGBs4 are remarkably inert organochlorine compounds usedfrom 1929 until the late 1970's in electrical systems, invest

ment casting, and numerous commercial products includingcarbonless copy paper and microscope immersion oil (7, 12).It has been estimated that only 4.4% of the PCBs purchasedby United States industry have been incinerated or degradedin the environment; the majority of PCBs are either still in use(60%), in land fills or dumps (23.5%), or free in the environment(11). Due to the resistance of PCBs to degradation, theirwidespread use has led to global contamination of the environment (5, 13, 14, 15). A 1972 report indicated that 32.5% ofrandomly collected samples of adipose tissue from the UnitedStates population contained 1.0 or greater ppm PCBs, 33.3%contained less than 1.0 ppm PCBs, and 34.2% contained nodetectable PCBs (46).

Biological effects of PCB exposure were suspected in 1968when Yusho, also known as "oil disease," reached epidemic

proportions in Japan due to widespread ingestion of rice oil

1This is Contribution 1115 of the Department of Biology, The Johns Hopkins

University. Supported in part by USPHS Contract C02664 (Stanford ResearchInstitute).

2 Supported in part by Public Health Service Training Grant 2 T32 GM 07231.3 Supported in part by USPHS Grant 1 R01 CA-26328-02 from the National

Cancer Institute. To whom requests for reprints should be addressed.4 The abbreviations used are: PCB, polychlorinated biphenyl; NCI, National

Cancer Institute; AP, alkaline phosphatase; H & E, hematoxylin and eosin.Received April 13. 1981; accepted August 24, 1981.

contaminated with PCBs (31). Symptoms of Yusho include eyedischarge, swelling of eyelids, acne-like skin eruptions, skin

pigmentation, and weakness (31).In experimental animals, some of the reported effects of

PCB-toxicity are liver alterations (chicken, mouse, guinea pig,

and rabbit) and skin lesions (guinea pig, rabbit, mouse, andmonkey) (1, 2, 4, 44). PCBs have been reported to inducehepatic smooth endoplasmic reticulum (38), hepatic enzymes(8, 32, 38), and clusters of pancreatic tissue in the livers (27)of exposed rats. Proliferative lesions in the liver, specificallyhepatocellular neoplastic nodules, have been reported in ratsfed Aroclor 1254 (24, 29, 43). Hepatocellular carcinomas havebeen reported in female Sherman rats fed Aroclor 1260 (30),and hepatomas have been induced in mice fed PCBs (23, 28).

Gastric lesions, such as epithelial hypertrophy, hyperplasia,and ulcÃ©ration,have been described in PCB-exposed monkeys(1, 2, 4, 34) and swine (20). Additionally, mucus-filled cyststhat penetrate the muscularis mucosa have been reported inrhesus monkeys exposed to PCBs (3, 6). In this report, wepresent evidence that Aroclor 1254 induces intestinal metaplasia in the glandular stomachs of F344 rats and that such lesionscan involve the muscularis mucosa and the submucosa andmay lead to adenocarcinoma.

MATERIALS AND METHODS

Bioassay. Male and female F344 rats (Simonsen Laboratory, Gilroy,Calif.) were fed a diet consisting of low-fat laboratory chow (Ralston

Purina Co., St. Louis, Mo.) that contained 0, 25, 50, or 100 ppmAroclor 1254 (CAS No. 27323-18-8; Lot KBO1 -604; Monsanto Chem

ical Company, St. Louis, Mo.) from July 1972 until September 1974.The test chemical was analyzed at Stanford Research Institute and wasfound to contain 54.67% chlorine and to be a mixture of at least 18isomers ranging from 4 to 7 chlorine atoms per molecule. Identificationor quantitation of impurities was not done. Aroclor 1254 was dissolvedin corn oil (Staley Manufacturing Co., Orange, Calif.) prior to its additionto the laboratory chow. Lungs, bronchi, spleen, liver, testes, pituitarygland, kidneys, brain, and any grossly visible lesions were preparedfor microscopy. In the initial studies performed at necropsy (43),stomachs from 42 control animals were examined histologically. Stomachs from PCB-treated animals were examined histologically only in

18 cases in which gross lesions were noted at necropsy. All tissueswere preserved in 10% buffered formalin, sealed in plastic bags, andstored at room temperature for 6 years at Stanford Research Instituteand the NCI Tissue Repository in Rockville, Md. Detailed informationon methods, results, and conclusions of the bioassay are available(43).

Detection of AP Activity in Formalin-fixed Specimens. Whole-tissue specimens of stomachs, which had been preserved in formalinfor 6 years, were washed overnight in deionized water and stained forAP activity, as described previously (35). Tissues were incubated atroom temperature for 3 hr in a solution of /8-naphthyl acid phosphate

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Arador 1254-induced Intestinal Metaplasia

(Sigma Chemical Company, St. Louis, Mo.), 0.005 M MgSO4-0.014

M sodium borate buffer (pH 10.1). Specimens were then drained anddipped into an aqueous solution of freshly prepared o-dianisidine (0.25

mg/ml) (Sigma Chemical Company). Development of a blue stain onthe duodenal cuff that was left attached to the specimen indicatedadequate staining. Specimens were examined under a dissecting microscope for AP activity in the stomach.

Histology. Following macroscopic localization of AP activity, tissueswere trimmed so that samples spanned the duodenum-glandular stomach junction and included AP-positive areas. Specimens were also

taken from the cardiac portion of the stomach. After embedding inparaplast-embedding medium (Lancer Company, St. Louis, Mo.), spec

imens were sectioned to thicknesses of 5 to 15 /im. Duplicate slideswere stained with H & E and with Alcian blue-periodic acid Schiffreagent. Selected slides were stained with phosphotungstic acid-he-matoxylin or eosin B-aniline blue (9), both of which stain Paneth cell

granules. Slides that were stored at the NCI Tissue Repository sincethe bioassay5 was performed 6 years ago were also reexamined.

RESULTS

Growth, General Health, and Survival. With the exceptionof males exposed to low doses of Aroclor 1254, the mean bodyweights of male and female rats fed Aroclor 1254 were lowerthan those of controls fed corn oil. Males and females fed thehigh dose had mean body weights that were roughly three-fourths and two-thirds those of controls, respectively. Begin

ning at Week 72 for rats fed the high dose and Week 104 forrats fed the medium dose, symptoms of Aroclor 1254 exposure,including alopecia, amber-colored urine, facial edema, exoph-

thalmos, and cyanosis, were evident (43). For males, 92% ofthe control group, 83% of the group exposed to the low dose,58% of the group exposed to the medium dose, and 46% ofthe group exposed to the high dose survived until the termination of the study. For females, 67% of the control group,79% of the group exposed to the low dose, 83% of the groupexposed to the medium dose, and 71 % of the group exposedto the high dose survived until the termination of the experiment(43).

Gross Morphology of the Stomachs. We first verified thatstomachs from the NCI Tissue Repository from both controland Aroclor 1254-treated rats had a normal gross morphology,except those from 18 Aroclor 1254-treated rats noted in the

original NCI report to have stomach tumors or other stomachlesions at necropsy (43). Specimens were then stained for APto pinpoint areas of possible pathological significance for histolÃ³gica! examination. In some specimens gross AP activitywas focal, while in others AP activity was diffuse and lessintense. The percentage of total specimens with focal AP was11 % in the control group, 13% in the group exposed to the lowdose, 27% in the group exposed to the medium dose, and 38%in the group exposed to the high dose of Aroclor 1254. DiffuseAP was present in 47% of the specimens from the controlgroup and greater than 75% of the specimens from each of theAroclor 1254-treated groups.

Sites of detectable AP activity overlapped only incompletelywith sites at which intestinal metaplasia or other pathologicallesions were detected by microscopic examination. When present, intestinal metaplasia was associated both with focal and

diffuse sites of AP activity. For specimens from the group fedthe high dose of Aroclor 1254, 10 of 16 (63%) pathologicallesions were at sites of focal AP activity, 4 of 16 (25%) were atregions of diffuse AP activity, and 2 of 16 (13%) were fromspecimens which had no detectable AP. One lesion scoredfrom an NCI tissue block was not included, since the portion ofthe stomach containing the lesion was not available for stainingfor AP activity.

HistolÃ³gica! Observations. All 191 stomachs available weresectioned through the junction of the pyloric region of thestomach and the duodenum, a known "hot spot" for gastric

neoplastic lesions in humans (36) and rats (33), as well as atany AP-positive sites. The incidence of microscopically con

firmed lesions in the glandular stomachs of F344 rats is presented in Table 1. The incidence of lesions in the stomachsincreased as a function of the concentration of Aroclor 1254 inthe diet, and at the highest concentration tested one-third of

the animals had gastric lesions. No significant differences wereapparent between males and females (Table 1). Multiple lesionswere found only in specimens from Aroclor 1254-treated rats

(Table 1).A histological classification of the types of gastric lesions

induced by Aroclor 1254 is presented in Table 2, and examplesof the lesions are shown in Figs. 1 to 12. Metaplastic lesions in3 controls were generally associated with aggregates of lym-

Table 1

Incidence of stomach lesions, namely intestinal metaplasia andadenocarcinoma, in the glandular stomachs of F344 rats exposed to Aroclor

1254Tissues were trimmed to include AP-positive sites as well as the junction

between the pyloric region of the stomach and the duodenum, embedded, andsectioned. Duplicate slides were stained with H & E and Alcian blue-periodic acidSchitf's reagent.

Concentration ofAroclor

1254(ppm)0

2550100No.

of maleswith stomachlesions2/24

(8.3)a

2/24 (8.3)4/24(16.7)7/24 (29.2)No.

of femaleswith stomach

lesions1/23

(4.3)3/24(12.5)4/24(16.7)

10/24(41.7)Rats

with multiple stomachlesions0/47

2/48 (4.2)1/48 (2.1)6/48(12.5)Total

rats withstomach lesions3/47

(6.4)5/48(10.4)8/48(16.7)

17/48(35.4)a Numbers in parentheses, percentages.

Table 2Histological analysis of stomach lesions0 in male and female F344 rats exposed

to Aroclor 1254 in their dietsTissues were trimmed to include AP-positive sites as well as the junction

between the pyloric region of the stomach and the duodenum, embedded, andsectioned. Duplicate slides were stained with H & E and Alcian blue-periodic acidSchiff's reagent.

IntestinalmetaplasiaConcen

tration ofAroclor

1254(ppm)025

50100CysticND"3

313Crypt-

type,over

lymph-oid clus

ter3

ND12Diffuse

in epitheliumND

11NDTotal3

45

15Adeno

carcinomaND

132Â°Total

ratswith

stomachlesions3

58

17Total

rats examined47

484848

5 The bioassay was performed at Stanford Research Institute, Menlo Park,

Calif, under the direction of Dr. David C. L. Jones. Necropsies were supervisedby Dr. Daniel Sasmore of Stanford Research Institute.

Rats containing multiple lesions were classified by the most severe lesion(see text).

6 ND. none detected.0 Regions of severe dysplasia were identified in 2 additional foci of intestinal

metaplasia, but sections available did not allow confirmation of the presence ofadenocarcinoma.

DECEMBER 1981 5053



fÃ¬.W. Morgan et al.

phocytes and/or inflammation (Fig. 1). Metaplastic cells werefew (Fig. 2). The histology of experimentally induced adeno-carcinoma of the rat glandular stomach has been described indetail (42). Most of the lesions were focal intestinal metaplasiacharacterized by mucin-containing goblet cells that stainedwith Alcian blue-periodic acid Schiff reagent (Figs. 3 to 6). Tall

columnar cells were commonly seen, and crypts of intestinalmetaplasia sometimes had atypical cells and mitotic figures(Fig. 4). Twenty-one of 33 lesions (64%) were adjacent to foci

of lymphocytes (Fig. 1), and some eosinophils and other inflammatory cells were noted. No Paneth cells were seen in slidesstained with H & E or in any of 14 lesions stained with phos-photungstic acid-hematoxylin or eosin B-aniline blue, which

specifically stained Paneth cell granules in specimens from therat ileum.

Nineteen specimens contained intestinal metaplasia organized into cysts (Figs. 3, 5, 7 to 10). Twelve cystic lesionsinvolved the muscularis mucosa, and 7 of the cysts distortedthe muscularis mucosa but did not actually appear to penetrateit. Three of the lesions in control rats, one in a rat exposed tothe medium dose of Aroclor 1254 and 2 from rats exposed tothe high dose of Aroclor 1254, were crypts of intestinal metaplasia located above large aggregates of lymphocytes (Figs. 1and 2). One lesion from a rat exposed to the low dose ofAroclor 1254 and one from a rat exposed to the medium doseconsisted of goblet cells scattered in the gastric epithelium butnot organized into intestinal crypts.

Adenocarcinomas were found in one specimen from thegroup exposed to the low dose of Aroclor 1254, in 3 specimensfrom rats exposed to the medium dose of Aroclor 1254, and in2 specimens from the group exposed to the high dose ofAroclor 1254 (Figs. 7 to 11). The carcinomas invaded themuscularis mucosa (1 case), the submucosa (4 cases), or theserosa (1 case) and contained areas resembling those ofintestinal metaplasia (Fig. 12) in addition to more undifferen-

tiated and scirrhous areas (Fig. 11).Twenty-nine of the 33 (88%) lesions scored were located in

the pyloric region of the stomach. Two of the lesions were inthe cardiac portion of the stomach, one of which was adjacentto the glandular stomach-forestomach junction. For 2 of the

lesions scored from slides stored at the NCI Tissue Repository,the locations were unknown.

Intestinal metaplasia was not seen in any of 8 animals thatdied before the 73rd week of the experiment. Because thenumber of animals that died during the first year of the experiment was so small, no conclusions can be drawn as to whenintestinal metaplasia first appeared. No correlation was foundbetween animals dying before the experiment ended at 105weeks and those having gastric lesions.

DISCUSSION

AP activity normally is not detectable in the rat glandularstomach (40) but is present at high levels in the brush bordersof the small intestine and persists after fixation of the tissues informalin at room temperature (17, 19, 41). Since intestinalmetaplasia and some adenocarcinomas contain AP, histo-

chemical staining for AP can be used to macroscopically pinpoint sites of possible pathological significance in stomachswhich have been preserved for extended periods of time (35).

A dose-dependent increase was seen in the incidence of

focal gastric AP in Aroclor 1254-treated animals. Although the

incidence of lesions was not 100% in animals exposed to thehigh dose, the high dose appeared to represent a maximallytolerated one. Diffuse AP activity was elevated in Aroclor 1254-treated animals, but the increase was not dose dependent.Diffuse AP activity was not seen in tissues from a similarbioassay in which rats were exposed to A/-methyl-A/'-nitro-/V-

nitrosoguanidine (35). Some areas of diffuse AP activity maystem from inflammation of the tissue or may reflect widespreadalteration of the epithelial cells of the gastric mucosa. Most ofthe stomachs with AP activity were histologically normal; however, 26 AP-positive specimens contained lesions of patholog

ical significance. Seven specimens which contained gastriclesions did not stain for AP, including 2 of the adenocarcinomas, 3 crypt-type lesions over lymphoid follicles, and 2 cystic

lesions (Table 2). Such lesions may represent a biochemicallydistinct category in that they lack AP. It has been reported thathuman intestinal metaplasia of the incomplete type lacks APactivity (26). An alternative explanation is that AP-negativegastric lesions contain AP at a level below the limit of detectionby macroscopic staining.

Intestinal metaplasia and total lesions in the glandular stomachs of F344 rats increased as a function of Aroclor 1254concentration in the diet, indicating that the lesions were eitherinduced or enhanced by exposure to the PCBs (Table 2). Insome cases, intestinal metaplasia disrupted the muscularismucosa, had features of adenocarcinoma, and resembledareas of invasive carcinomas. A definite sequence of cancerdevelopment from these metaplastic areas, however, could notbe demonstrated.

Stomach adenocarcinomas are rarely found in aging F344rats. In untreated F344 rats used as controls in carcinogenesistests performed as part of the NCI's Carcinogenesis Testing

Program, 1 of 1754 (0.05%) stomach adenocarcinomas wereobserved in females and 0 of 1794 (<0.05%) were observedin males (18). The initial pathological analysis performed atStanford Research Institute detected one adenocarcinoma inan animal from the low-dose groups and 2 in animals from themedium-dose groups. Our study has detected 3 additionalearly adenocarcinomas at sites of AP-positive intestinal meta

plasia (Table 2) as well as 2 foci of severe intestinal metaplasiain which adenocarcinoma could not be identified with certainty(Table 2, Footnote c). Using simultaneous controls in which thefrequency of stomach adenocarcinomas is less than 2% (<1 of47) and assuming that the occurrence of adenocarcinoma israndom, the likelihood of observing 6 adenocarcinomas in 144rats is less than 0.05 (significant at p < 0.05). With the use ofhistorical controls in which the incidence of adenocarcinomasof the stomach is less than 0.05% (1 of 3548) and assumingthat the occurrence of adenocarcinoma is random, the chanceof observing 6 adenocarcinomas in 144 rats is less than 1.3x 10~10 (significant at p < 0.001 ).

Over one-half of the gastric lesions were adjacent to aggre

gates of lymphocytes. Similar clusters were found in areaslacking AP staining or proliferative lesions in the stomachs oftreated animals. The lymphoid aggregates may be folliclesnormally present in the stomach, although we detected no suchstriking clusters in control animals in this or an earlier experiment (35). It is also possible that the lymphoid aggregates stemfrom an immune response to inflammation of the gastric mucosa or to altered cellular antigens. Although striking when




Aroclor 1254-induced Intestinal Metaplasia

present, lymphoid clusters are not obligatory in the development of intestinal metaplasia since some sites of intestinalmetaplasia lacked discernible lymphoid aggregates.

Individual animal pathology data were examined in an attempt to uncover any correlation between rats having stomachlesions and those with liver lesions, namely, hepatocellularcarcinomas and hepatocellular neoplastic nodules. Hepatocellular neoplastic nodules can be induced by chemical carcinogens and increasingly are recognized as "precancerous" le

sions (16, 39, 45). No correlation was found between animalshaving gastric lesions and those with liver lesions; therefore,the presence of intestinal metaplasia does not appear to predispose an animal to "precancerous" lesions in the liver, or

vice versa.The presence of intestinal enzymes and cell types in gastric

foci may be significant in stomach cancer progression (21, 26).Although it remains to be established that intestinal metaplasiais "precancerous," intestinal metaplasia accompanies stom

ach cancer (25, 36), and both carcinoma and intestinal metaplasia are found in the pyloric region of the stomach (37). Theincidences of intestinal metaplasia and carcinoma increasewith age, and populations with high frequencies of gastriccancer have increased incidences of intestinal metaplasia compared to those with low frequencies of gastric cancer (10, 22).Furthermore, in this and in a previous study (35), we havefound early carcinomas preferentially located at sites of intestinal metaplasia. Thus, adenocarcinoma and intestinal metaplasia appear to be related and may share initiating mechanisms.Our results indicate that evaluation of data on intestinal metaplasia can increase the sensitivity of an animal bioassay fordetecting gastric carcinogens and/or promoters.

The conclusion from the NCI bioassay was that the combinedincidence of hepatocellular neoplastic nodules and hepatocellular carcinomas in animals treated with Aroclor 1254 wassignificantly higher than that in controls. We have extended theresults from this bioassay and conclude that Aroclor 1254certainly leads to induction of intestinal metaplasia and probably leads to induction of adenocarcinoma in the glandularstomachs of F344 rats.

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R. W. Morgan et al.

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Fig. 1. Glandular stomach of control female F344 rat showing a focus of hyperplastic glands within lymphoid aggregate. Note disruption of muscularis mucosa.H & E, X 54.

Fig. 2. Alcian blue-periodic acid-Schiff staining of lesion in Fig. 1. A few mucus cells are present, x 130.

Fig. 3. Focal intestinal metaplasia in glandular stomach of rat receiving Aroclor 1254. Note fibrosis and inflammation adjacent to the lesion. H & E, x 80.

Fig. 4. Portion of gastric intestinal metaplasia in Fig. 3 showing mitotic figures, hyperplasia, goblet cells, and tall columnar cells. Note inflammatory cells in laminapropria. H & E. x 220.

DECEMBER 1981 5057



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8Fig. 5. Alcian blue-periodic acid-Schiff staining of lesion in Fig. 3 showing goblet cells and mucus in lesion but not in adjacent normal epithelium, x 68.

Fig. 6. Alcian blue-periodic acid-Schiff staining of lesions in Figs. 3 and 5 showing Alcian blue-positive goblet cells and periodic acid-Schiff-positive brush border.x. 220.

Fig. 7. Portion of hyperbasophilic focus of poorly differentiated intestinal metaplasia. Note similarity to invasive adenocarcinoma. H & E, x 54.

Fig. 8. Gastric adenocarcinoma invading the tunica muscularis. Note ulcÃ©rationand inflammation. H & E, x 54.

5058



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Fig. 9. Portion of large metaplastic lesion showing apparent early invasion into submucosa. H & E, x 54.

Fig. 10. Portion of large cystic gastric submucosal lesion with similarities to intestinal metaplasia and adenocarcinoma. H & E. x 55.

Fig. 11. Scirrhous gastric adenocarcinoma which invaded to the gastric serosa. H & E. x 80.Fig. 12. Portions of gastric adenocarcinoma which invaded the gastric serosa showing areas of well-differentiated epithelial cells resembling those in intestinal

metaplasia. H & E, x 220.

5059



1981;41:5052-5059. Cancer Res Robin W. Morgan, Jerrold M. Ward and Philip E. Hartman Adenocarcinoma in the Glandular Stomach of F344 RatsAroclor 1254-induced Intestinal Metaplasia and

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