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Ethyl acrylate-induced gastric toxicity
roxIcomcy
AND
APPLIED
83,576-583
PHARMACOLOGY
(1986)
Ethyl Acrylate-Induced III. Development B. 1. GHANAYEM,’ National
Tosicology
and Recovery
November
B. I., MARONPOT,
Gastric
5. 1985; accepted
Toxicity.
Health Sciences, National North Carolina 27709
January
Institutes
of‘ Health,
10. 1986
and Recovery of Lesions. GHANTouicol. Appl. Pharmacol. 83, 576-583. Rats receiving 14 daily gavage doses of 100 or 200 mg/kg ethyl acrylate (EtAc) and killed at varying times following the end of dosing exhibited dose-dependent lesions and recovery from lesions in the forestomach. The glandular stomach which was previously shown to be affected by acute exposure to EtAc appeared to have adapted to resist EtAc toxicity with repeat exposure and appeared normal in all animals. Adaptation of the forestomach was characterized by increased papillomatous thickening with dose. Lesions observed in acute exposure to EtAc were stil present with repeat dosing and were more pronounced at the high dose. Forestomachs of rats which received 100 mg/kg EtAc for 14 days were recovered to normal within 2 weeks following the last dose. Forestomachs of rats receiving 200 mg/kg EtAc still exhibited numerous lesions 2 weeks following the last dose, and mucosal hyperplasia was present in the forestomachs at 4 weeks postexposure. Two lesions. submucosal fibrosis and foreign body reaction, became more prevalent in high-dose animals with time. Foreign body reaction, which was present in all animals 4 weeks postexposure, appeared to have resulted from entrapment of hair and/or feed particles in forestomach lesions in the course of healing. It is speculated that the increased cell proliferation and the induced foreign body reactions may contribute to the previously demonstrated carcinogenic effect of EtAc on the rat forestomach. 0 1986 Academic Press, Inc.
AYEM,
Acrylate-Induced
of Lesions
R. R. MARONPOT, AND H. B. MATTHEWS
Program, National Institute of‘Environmenfa1 P.O. BO.Y 12233, Research Triangle Park.
Received
Ethyl
Gastric Toxicity
R. R., AND
III. Development
MATTHEWS,
Esters of acrylic acid have widespread industrial and consumer product applications. Most common uses are as polymers and copolymers in latex paints, paper coatings, fabric finishes, dirt release agents, and specialty plastics (IARC, 1979). In addition, polymers of ethyl acrylate are approved as components of food packaging products (U.S. CFR, 1978). The toxicity of ethyl acrylate has been documented in inhalation (Miller d al., 1980), oral (Ghanayem ei al., 1985a), and dermal (Hengler and DePass, 1982) studies. Results of these studies indicate that ethyl acrylate is an irritating chemical producing lesions in the tissues directly exposed to the chemical. In in’ To whom 0041-008X/86
correspondence
should
$3.00
Copyngbt 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
be addressed 576
H. B. (1986).
halation studies, the tissues most severely affected are in the nasal cavity. In gavage studies, the forestomach is the primary target tissue. The epidermis and dermis are the primary target tissues when exposure is by skin painting. While compound-related pathologic changes have been described in other internal organs, these changes are nonspecific and are observed primarily in animals that die during the course of treatment. Long-term inhalation (Miller rf al.. 1985) and gavage (NTP, 1983) studies have been undertaken to ascertain the carcinogenicity of ethyl acrylate. Ethyl acrylate (EtAc) was found to be carcinogenic in rats and mice when administered by gavage in corn oil (NTP, 1983) but was not carcinogenic when exposure was
ETHYL
ACRYLATE-INDUCED
by inhalation (Miller et al., 1985) or by dermal application (Hengler and DePass, 1982). In the chronic gavage study, EtAc doses similar to those used in the current study were carcinogenic, causing squamous cell papillomas and carcinomas of the forestomach in both sexes of F344/N rats and B6C3FI mice (NTP, 1983). Previously, the pathogenesis and mechanisms associated with the early acute gastric lesions induced by EtAc have been characterized in this laboratory (Ghanayem et al., 1985a,b). These studies demonstrated that gastric toxicity is both dose and time dependent. The present study addresses the gastric toxicity of repeat doses of EtAc (2 weeks) administered at the same doses to the same strain of rats used in the NTP chronic toxicity study and examines the reversibility of the EtAc-induced gastric lesions.
GASTRIC
TOXICITY
577
the last LOO-mg/kg dose of EtAc and 1, 14, or 28 days after the last 200-mg/kg dose of EtAc. At the time of termination rats were anesthetized with ether, an abdominal incision was made and the entire stomach was removed and processed for histopathological examination as previously described (Ghanayem et a/., 1985a); pathologic evaluation was performed without prior knowledge of treatment.
RESULTS The effect of 14 consecutive daily doses of EtAc, 100 and 200 mg/kg, on body weight gain (expressed as percentage of starting weight) is shown in Fig. 1. There was no significant effect of 100 mg/kg EtAc on body weight gain when treated rats were compared to vehicle control rats. Gavage administration of 200 mg/kg EtAc resulted in significant depression of body
METHODS dnimals and animal treatment. Male Fischer 344 rats obtained from Charles River Breeding laboratory (Wilmington, Mass.) were used throughout the current studies. They were housed in polycarbonate cages (four/cage) in facilities with an ambient temperature of 2 I to 22°C. relative humidity of 50 + lo%, and a 12-hr dark-light cycle. They were given NIH 3 1 diet and water ad libitum. Rats were conditioned in our facilities for a minimum of 5 days after arrival from the breeder and prior to use in our experiments. All animals used weighed between 200 and 230 g. Ethyl acrylate (99%; inhibited with 15 to 20 ppm of hydroquinone monomethyl ether, Lot 022 1MJ) was purchased from Aldrich Chemical Company (Milwaukee, Wise.). The desired dose of EtAc was dissolved in Mazola corn oil (dose volume = 5 ml/kg) and given via a stainlesssteel feeding tube by gavage. Control rats were given the same volume of corn oil by gavage. Treatment times (lO:OO-12:00 AM) were standardized to be the same for all groups. The dosing solutions were prepared weekly and stored at -20°C. EtAc dissolved in corn oil was previously shown to be stable for 8 days (NTP, 1983). Three groups of rats were treated as follows: Group 1 (24 rats) was treated daily with 5 ml corn oil/kg for 14 consecutive days. Group 2 (28 rats) was treated daily with 100 mg EtAc/S ml corn oil/kg for 14 consecutive days. Group 3 (28 rats) was treated daily with 200 mg EtAc/5 ml corn oil/kg for 14 consecutive days. Each group was divided into subgroups and killed 1, 7, or 14 days after
TIME (weeks)
FIG. 1. Effect of daily gavage administration of corn oil (A), 100 mg/kg (0). and 200 mg/kg (m) EtAc on body weight gain of rats treated for 14 consecutive days. Values are the X i SE (a) values which are significantly lower than control; (b) values which are significantly lower than the 100 mg/kg values at p G 0.05.
578
GHANAYEM,
MARONPOT,
weight gain during the 2 weeks of treatment (Fig. 1). However, by 2 weeks after the last of 14 consecutive daily doses of 200 mg/kg EtAc, body weight gain was not different from that of vehicle controls or 100 mg/kg EtAc-treated rats (Fig. 1). Glandular stomach lesions observed in earlier studies after gavage administration of one to four daily doses of EtAc ( 100 or 200 mg/ kg) to male F344 rats (Ghanayem et al., 1985a) were not observed in rats treated with the same doses of EtAc for 14 consecutive days (data not shown).
FIG. 2. Gross appearance of the gavage for 14 consecutive days. (a) 200 mg/kg, showing papillomatous same lesions as in (c) as well as hair
AND
MATTHEWS
The effect of 14 daily doses of EtAc on the rat forestomach was readily apparent at necropsy (Fig. 2). Forestomachs of rats given 100 mg/kg were slightly thickened, whereas severe papillomatous thickening characterized the forestomachs of rats treated with 200 mg/kg EtAc. Hair shafts and feed particles were entrapped in the forestomach mucosa of rats given 200 mg EtAc/kg. Daily gavage administration of 100 mg/kg or 200 mg/kg EtAc to rats also caused microscopic lesions in the forestomachs (Table 1). In the 100 mg/kg EtAc-treated group, histo-
mucosal surface of formalin-fixed stomachs from rats given EtAc by Control; (b) 100 mg/kg, showing slight enlargement of the stomach; (c) thickening and folding of the forestomach; (d) 200 mg/kg showing the shafts embedded in the forestomach.
ETHYL
ACRYLATE-INDUCED
GASTRIC
TABLE INCIDENCE
Lesion Mucosal ulceration Mucosal hyperplasia Mucosal inflammation Hyperkeratosis Submucosal intlammation Foreign body reaction Submucosal fibrosis ’ Ethyl daily dose in groups b Time
1
OF HISTOPATHOLOGIC LESIONS IN THE FORESTOMACH OF RATS GIVEN AND KILLED AT VARYING TIMES AFTER THE LAST IXXE EtAc Control O/24 O/24 O/24 O/24 O/24 O/24 O/24
lb o/12 12112 o/12 12112 o/12 o/12 o/12
579
TOXICITY
EtAc’
EtAc
100 mg/kg I
O/8 618 O/8 618 018 O/8 O/8
FOR 14 DAYS
200 mg/kg
14
lb
14
018 018 018 018 018 018 I/8
8/12 12112 l/l2 12112 12112 o/12 o/12
O/8 818 3/g
w3 3/g
l/8 l/8
28 O/8
8/8 O/8 O/8 018 w3 318
acrylate was dissolved in corn oil to standardize dose volume at 5 ml/kg body weight, and the appropriate was given by gavage for 14 consecutive days. Control rats received 5 ml/kg corn oil alone and were killed with the corresponding EtAc-treated rats. (days) of termination after the last of 14 consecutive daily doses of EtAc.
pathologic changes in the forestomach consisted of marked generalized hyperkeratosis and moderate to marked generalized hyperplasia of the stratified squamous cell mucosal layer (Fig. 3 and Table 1). These and additional lesions were observed in the forestomach of rats which were given 200 mg/kg EtAc. Eight of twelve rats treated with 200 mg/kg EtAc had multiple mucosal ulcers with attendant purulent inflammation in adjacent mucosal epithelial layers. While there was marked generalized submucosal inflammation, this change was most severe in the vicinity of the mucosal ulcers (Fig. 3). The submucosal inflammation was characterized by infiltraton of neutrophils and lymphocytes and by early fibrosis (Fig. 3). All intact mucosal surfaces were hyperplastic and were covered by a thick layer of keratin (Fig. 3). One week after the last of 14 consecutive daily doses of 100 mg/kg EtAc, there was a decline in the incidence of the mucosal hyperplasia and hyperkeratosis of the forestomsch. These lesions completely disappeared by 2 weeks after the last dose (Table 1). Submucosal fibrosis was observed in one of eight rats 2 weeks after the last of 14 EtAc doses ( 100 mg/kg). In rats treated with 200 mg/kg EtAc for 14 consecutive days, there was a
complete disappearance of forestomach mucosal ulceration and a significant decline in the incidences of mucosal and submucosal inflammation within 2 weeks after the last dose (Table 1). Acute generalized inflammation (both mucosal and submucosal) disappeared within 4 weeks after the last 200-mg/kg EtAc dose. In contrast, the incidence of mucosal hyperplasia of the forestomach was unchanged 4 weeks after the last 200-mg/kg EtAc dose (Table 1). Hyperkeratosis was not observed in any animals at the end of this same recovery period. Two additional histologic changes were observed at increased incidence in the forestomachs of high dose rats at the end of the 4-week recovery period. Fibrosis, characterized by deposition of mature collagen, caused a thickening of the submucosa in three of eight rats given 200 mg/kg EtAc (Table 1 and Fig. 4). The second histologic change was multifocal foreign body reaction to embedded hair shafts and was present in the submucosa of all eight rats that had received 200 mg/kg EtAc (Table 1 and Fig. 5). The mucosa overlying these foreign body reactions was more severely hyperplastic than the adjacent mucosa. Similar changes were observed in only one rat following a 2-week recovery period.
GHANAYEM.
MARONPOT,
AND
MATTHEWS
a
of forestomachs from rats given EtAc by gavage for 14 consecutive days. (a) FIG. 3. Photomicrographs Control; (b) 100 mg/kg, showing hyperplasia and hyperkeratosis of the mucosal epithelium; (c) 200 mg/kg, showing mucosal ulceration (upper left), papillomatous hyperplasia of the mucosa (upper right), and severe submucosal inflammation. Hematoxylin and eosin stain. X I 15 (a and b) and X40 (c).
DISCUSSION Chronic gavage administration of 100 or 200 mg/kg EtAc to F344 rats (5 days/week for 2 years) caused hyperkeratosis, hyperplasia, squamous cell papillomas, and squamous cell carcinomas of the forestomach (NTP, 1983).
In subsequent studies (Ghanayem et al., 1985a), we demonstrated that gavage administration of similar doses of EtAc to F344 male rats caused acute lesions in both the forestomach and the glandular stomach. The present studies were conducted to expand upon our previous studies by characterizing gastric le-
FIG. 4. Submucosal layer thickened by mature fibrous connective tissue of rats treated with 14 consecutive daily doses of 200 mg/kg EtAc and killed 4 weeks after the last EtAc dose. Overlying epithelium hyperplastic. Hematoxylin and eosin. X64.
FIG. 5. A foreign body inflammatory reaction surrounds a hair shaft (arrow) embedded of rats treated with 14 consecutive daily doses of 200 mg/kg EtAc and killed 4 weeks dose. The overlying epithelium is hyperplastic. Hematoxylin and eosin. X53. 581
in the submucosa after the last EtAc
582
GHANAYEM.
MARONPOT.
sions induced by repetitive exposure to EtAc and by investigating the reversibility of EtAcinduced gastric lesions. Evidence of gastric toxicity was present in the forestomach of all rats treated with 14 consecutive daily gavage dosesof EtAc. However, EtAc-induced glandular stomach lesionsobserved after up to four consecutive daily dosesof EtAc (Ghanayem et al., 1985a) were not present following 14 consecutive daily gavage doses.Therefore, it appearsthat the glandular stomach recovered from acute effects and adapted to the effects of repeat exposure to EtAc with no persistent morphologic alterations. The forestomach, which was more sensitive to both the acute (Ghanayem et al., 1985a,b) and chronic effects (NTP, 1983) of EtAc, was also shown in the current studies to be more sensitive than the glandular stomach to repeated EtAc dosing ( 14 daily doses).Mucosal hyperplasia and hyperkeratosis of the forestomach were observed in all rats treated with 14 daily dosesof 100 or 200 mg/kg EtAc. In addition, mucosal ulceration as well as mucosal and submucosal inflammation were observed only in high-dose rats (200 mg/kg). Forestomach lesions in rats receiving EtAc were more numerous and more severe in the high-dose animals. This may explain the subsequent reversibility of EtAc-induced forestomach lesionsduring the recovery period in the low doserats versusthe persistenceof some of the alterations in the high dose rats (Table 1). Mucosal hyperplasia, which disappearedin animals receiving the low dose of EtAc, was lessseverebut still present in all animals killed 4 weeks following the last 200-mg/kg dose (Table 1). However, two lesions, submucosal fibrosis and foreign body reaction, became more prevalent in animals receiving the high doseas the recovery period progressed(Table I). The foreign body reaction and submucosal fibrosis represent lesions which were not observed at the end of the dosing regimen and must have developed during the recovery period. Furthermore, the foreign body reactions were apparently due to broken hair shaftsand/
AND
MATTHEWS
or feed particles entrapped in the forestomach mucosa during the healing process. What role, if any, the occurrence of foreign bodies(hair shaftsand/or feed particles) in the forestomachsof ethyl acrylate-treated rats may have in the subsequentdevelopment of benign and malignant forestomach tumors is unknown. However, experimental foreign body carcinogenesisis well establishedin the urinary bladder (Clayson and Pringle, 1966; Clayson et al., 1967) and subcutis (Farber and Cameron, 1980) of rodents, and potential counterparts in human cancer development include gallbladder tumors secondary to cholesterol stones, urinary bladder tumors secondary to chronic schistosomiasis,and lung tumors associated with asbestos(Farber and Cameron, 1980). It is conceivable that the foreign bodies observedin the present study might contribute to the forestomach tumor burden observed in the 2-year carcinogenesisstudy (NTP, 1983). We have demonstrated here that repetitive dosing with EtAc induces persistent proliferative changesin the forestomach of rats. It has been statedthat “induction of abnormally high levels of cell proliferation in a tissue provides a favorable environment for the development of cancer” (Iverson et ul., 1985). Experimental evidence that cellular proliferation secondary to injury is associatedwith enhanced susceptibility to chemical carcinogenesisat the site of cell proliferation hasbeen demonstrated for the glandular stomach (Nagai et al., 1984) as well as for the skin (Argyris and Slaga, 1981: Argyris, 1980). Similar observations have been made for liver (Pound and McGuire, 1978), urinary bladder (Clayson and Pringle, 1966) and mammary gland (Nandi, 1978). It is tempting to speculate that this phenomenon may be operational in the forestomach of rats given repeated gavage dosesof ethyl acrylate. Entrapment of foreign bodies and resultant reaction in the course of forestomach healing may serve to further exacerbate EtAc toxicity and cell proliferation. Further, EtAc is mutagenic in some systems(NTP, 1983) and increasedcell proliferation in its presencewould increasethe amount of replicating DNA at risk
ETHYL
ACRYLATE-INDUCED
for mutagenic alteration. Therefore, one or more of these factors alone or in combination may account for EtAc carcinogenicity to rat forestomach. Tissue changes, cell proliferation, foreign body reactions, and carcinogenicity are much more pronounced at EtAc doses of 200 mg/kg than at 100 mg/kg. Therefore, it may be that doses which produce increased and persistent cell proliferation and foreign body reactions would also be carcinogenic. REFERENCES ARGYRIS. T. S. (1980). Tumor promotion by abrasion induced epidermal hyperplasia in the skin of mice. J. Inwt. Dermatol. 75, 360-362. ARGYRIS. T. S., AND SLACA, T. J. (1981). Promotion of carcinomas by repeated abrasion in initiated skin of mice. Cancer ReJ. 41, 5193-5195. CLAYSON. D. B., AND PRINGLE, J. A. S. (1966). The influence of a foreign body on the induction of tumours in the bladder epithelium of the mouse. Brit. J. Cancer 20, 564-568. CLAYSON, D. B., PRINGLE. J. A. S.. AND BONSER.G. M. ( 1967). 4-Ethylsulphonylnaphthalene- I-sulphonamide: A new chemical for the study of bladder cancer in the mouse. Biochem. Pharmacol. 16, 619-626. FARBER, E., AND CAMERON, R. (1980). The sequential analysis of cancer development. In Advances in Cancer Research (F. C. Austin and C. W. Boone, eds.), Vol 3 I, pp. 125-226. Academic Press, New York. GHANAYEM, G. I., MARONPOT, R. R.. AND MATTHEWS, H. B. (1985a). Ethyl acrylate-induced gastric toxicity. I. Effect of single and repetitive dosing. Toxicol. Appl. Pharmacol. 80, 323-335. GHANAYEM, B. 1. MARONPOT. R. R., AND MATTHEWS, H. B. (1985b). Ethyl acrylate-induced gastric toxicity. II. Structure toxicity relationships and mechanism. To.uicol. .4pp,1.Pharmacol. 80, 336-344.
GASTRIC
583
TOXICITY
HENGLER, W. C.. AND DEPASS, L. R. (1982). Eth.yl Acrylate: Lifetime Dermal Carcinogenesis Stud?) in C3H/ HEJ Male Mice. Project Report 45-5 13. Union Carbide Corporation Bushy Run Research Center. International Agency for Research on Cancer (IARC) (1979). Some monomers, plastics and synthetic elastomers. and acrolein. ZARC Monogr. Eva/. Carcirwg. Risk Chem. Man 19, 57-62. IVERSON, F.. LOK, E., RENA, E., KARPINSKI. K., AND CLAYSON, D. B. (1985). A 13-week feeding study of butylated hydroxyanisole: The subsequent regression of the induced lesions in male Fischer F344 rat forestomach epithelium. Toxicolog.v 35, I- 1 I. MILLER, R. R.. AYERS, J. A.. RAMPY, L. W., SMITH. J. M., WEIL, C. S., CLARY, J. J.. AND JERSEY,G. D. (1980). Ethyl acrylate vapor inhalation study: 3-Month sacrifice of rats and 6-month sacrifice of rats and mice (abstract 159). In 19th .4nnua/ Society of Tkcolo,q Meeting, RhPhington, D.C. MILLER. R. R.. YOUNG, J. T.. KOCIBA. R. J.. KEYES, D. G.. BODNER. K. M.. CALHOUN, L. L., AND AYRES. J. A. (1985). Chronic toxicity and oncogenicity bioassay of inhaled ethyl acrylate in Fischer 344 rats and B6C3FI mice. Drug Chem. To\-icol. 8, l-42. NAGAI,
T., FFEIFFER,
C. J., FUJIMURA,
M.,
HATTORI.
T.,
TOBE. T. ( 1984). Susceptibility of healed gastric ulcers to chemical carcinogenesis in rats and implications of cellular kinetic changes. Canc~erRex 44, 5828-5835. NANDI, S. (1978). Hormonal carcinogenesis: A novel hypothesis for the role of hormones. J. Environ. Pathol. Toricol. 2, 13-20. National Toxicology Program (NTP) (1983). Carcino~qw esis Bioassa~~(!I Ethyl, lcrylate. ‘Technical Report Series 259, Publication (NIH) 82-25 15. U. S. Department of Health and Human Services. Public Health Service, National Institutes of Health. Research Triangle Park N. C. POUND. A. W.. AND MCGUIRE, L. J. (I 978). Influence of repeated liver regeneration on hepatic carcinogenesis by diethylnitrosamine in mice. Bri/. J. Cancer 37, 595602. U.S. CFR (1978). L:.S. Code qf.b>deral Regulatrons 21. 175-210. 176-180. 181-230. AND
AND
APPLIED
83,576-583
PHARMACOLOGY
(1986)
Ethyl Acrylate-Induced III. Development B. 1. GHANAYEM,’ National
Tosicology
and Recovery
November
B. I., MARONPOT,
Gastric
5. 1985; accepted
Toxicity.
Health Sciences, National North Carolina 27709
January
Institutes
of‘ Health,
10. 1986
and Recovery of Lesions. GHANTouicol. Appl. Pharmacol. 83, 576-583. Rats receiving 14 daily gavage doses of 100 or 200 mg/kg ethyl acrylate (EtAc) and killed at varying times following the end of dosing exhibited dose-dependent lesions and recovery from lesions in the forestomach. The glandular stomach which was previously shown to be affected by acute exposure to EtAc appeared to have adapted to resist EtAc toxicity with repeat exposure and appeared normal in all animals. Adaptation of the forestomach was characterized by increased papillomatous thickening with dose. Lesions observed in acute exposure to EtAc were stil present with repeat dosing and were more pronounced at the high dose. Forestomachs of rats which received 100 mg/kg EtAc for 14 days were recovered to normal within 2 weeks following the last dose. Forestomachs of rats receiving 200 mg/kg EtAc still exhibited numerous lesions 2 weeks following the last dose, and mucosal hyperplasia was present in the forestomachs at 4 weeks postexposure. Two lesions. submucosal fibrosis and foreign body reaction, became more prevalent in high-dose animals with time. Foreign body reaction, which was present in all animals 4 weeks postexposure, appeared to have resulted from entrapment of hair and/or feed particles in forestomach lesions in the course of healing. It is speculated that the increased cell proliferation and the induced foreign body reactions may contribute to the previously demonstrated carcinogenic effect of EtAc on the rat forestomach. 0 1986 Academic Press, Inc.
AYEM,
Acrylate-Induced
of Lesions
R. R. MARONPOT, AND H. B. MATTHEWS
Program, National Institute of‘Environmenfa1 P.O. BO.Y 12233, Research Triangle Park.
Received
Ethyl
Gastric Toxicity
R. R., AND
III. Development
MATTHEWS,
Esters of acrylic acid have widespread industrial and consumer product applications. Most common uses are as polymers and copolymers in latex paints, paper coatings, fabric finishes, dirt release agents, and specialty plastics (IARC, 1979). In addition, polymers of ethyl acrylate are approved as components of food packaging products (U.S. CFR, 1978). The toxicity of ethyl acrylate has been documented in inhalation (Miller d al., 1980), oral (Ghanayem ei al., 1985a), and dermal (Hengler and DePass, 1982) studies. Results of these studies indicate that ethyl acrylate is an irritating chemical producing lesions in the tissues directly exposed to the chemical. In in’ To whom 0041-008X/86
correspondence
should
$3.00
Copyngbt 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
be addressed 576
H. B. (1986).
halation studies, the tissues most severely affected are in the nasal cavity. In gavage studies, the forestomach is the primary target tissue. The epidermis and dermis are the primary target tissues when exposure is by skin painting. While compound-related pathologic changes have been described in other internal organs, these changes are nonspecific and are observed primarily in animals that die during the course of treatment. Long-term inhalation (Miller rf al.. 1985) and gavage (NTP, 1983) studies have been undertaken to ascertain the carcinogenicity of ethyl acrylate. Ethyl acrylate (EtAc) was found to be carcinogenic in rats and mice when administered by gavage in corn oil (NTP, 1983) but was not carcinogenic when exposure was
ETHYL
ACRYLATE-INDUCED
by inhalation (Miller et al., 1985) or by dermal application (Hengler and DePass, 1982). In the chronic gavage study, EtAc doses similar to those used in the current study were carcinogenic, causing squamous cell papillomas and carcinomas of the forestomach in both sexes of F344/N rats and B6C3FI mice (NTP, 1983). Previously, the pathogenesis and mechanisms associated with the early acute gastric lesions induced by EtAc have been characterized in this laboratory (Ghanayem et al., 1985a,b). These studies demonstrated that gastric toxicity is both dose and time dependent. The present study addresses the gastric toxicity of repeat doses of EtAc (2 weeks) administered at the same doses to the same strain of rats used in the NTP chronic toxicity study and examines the reversibility of the EtAc-induced gastric lesions.
GASTRIC
TOXICITY
577
the last LOO-mg/kg dose of EtAc and 1, 14, or 28 days after the last 200-mg/kg dose of EtAc. At the time of termination rats were anesthetized with ether, an abdominal incision was made and the entire stomach was removed and processed for histopathological examination as previously described (Ghanayem et a/., 1985a); pathologic evaluation was performed without prior knowledge of treatment.
RESULTS The effect of 14 consecutive daily doses of EtAc, 100 and 200 mg/kg, on body weight gain (expressed as percentage of starting weight) is shown in Fig. 1. There was no significant effect of 100 mg/kg EtAc on body weight gain when treated rats were compared to vehicle control rats. Gavage administration of 200 mg/kg EtAc resulted in significant depression of body
METHODS dnimals and animal treatment. Male Fischer 344 rats obtained from Charles River Breeding laboratory (Wilmington, Mass.) were used throughout the current studies. They were housed in polycarbonate cages (four/cage) in facilities with an ambient temperature of 2 I to 22°C. relative humidity of 50 + lo%, and a 12-hr dark-light cycle. They were given NIH 3 1 diet and water ad libitum. Rats were conditioned in our facilities for a minimum of 5 days after arrival from the breeder and prior to use in our experiments. All animals used weighed between 200 and 230 g. Ethyl acrylate (99%; inhibited with 15 to 20 ppm of hydroquinone monomethyl ether, Lot 022 1MJ) was purchased from Aldrich Chemical Company (Milwaukee, Wise.). The desired dose of EtAc was dissolved in Mazola corn oil (dose volume = 5 ml/kg) and given via a stainlesssteel feeding tube by gavage. Control rats were given the same volume of corn oil by gavage. Treatment times (lO:OO-12:00 AM) were standardized to be the same for all groups. The dosing solutions were prepared weekly and stored at -20°C. EtAc dissolved in corn oil was previously shown to be stable for 8 days (NTP, 1983). Three groups of rats were treated as follows: Group 1 (24 rats) was treated daily with 5 ml corn oil/kg for 14 consecutive days. Group 2 (28 rats) was treated daily with 100 mg EtAc/S ml corn oil/kg for 14 consecutive days. Group 3 (28 rats) was treated daily with 200 mg EtAc/5 ml corn oil/kg for 14 consecutive days. Each group was divided into subgroups and killed 1, 7, or 14 days after
TIME (weeks)
FIG. 1. Effect of daily gavage administration of corn oil (A), 100 mg/kg (0). and 200 mg/kg (m) EtAc on body weight gain of rats treated for 14 consecutive days. Values are the X i SE (a) values which are significantly lower than control; (b) values which are significantly lower than the 100 mg/kg values at p G 0.05.
578
GHANAYEM,
MARONPOT,
weight gain during the 2 weeks of treatment (Fig. 1). However, by 2 weeks after the last of 14 consecutive daily doses of 200 mg/kg EtAc, body weight gain was not different from that of vehicle controls or 100 mg/kg EtAc-treated rats (Fig. 1). Glandular stomach lesions observed in earlier studies after gavage administration of one to four daily doses of EtAc ( 100 or 200 mg/ kg) to male F344 rats (Ghanayem et al., 1985a) were not observed in rats treated with the same doses of EtAc for 14 consecutive days (data not shown).
FIG. 2. Gross appearance of the gavage for 14 consecutive days. (a) 200 mg/kg, showing papillomatous same lesions as in (c) as well as hair
AND
MATTHEWS
The effect of 14 daily doses of EtAc on the rat forestomach was readily apparent at necropsy (Fig. 2). Forestomachs of rats given 100 mg/kg were slightly thickened, whereas severe papillomatous thickening characterized the forestomachs of rats treated with 200 mg/kg EtAc. Hair shafts and feed particles were entrapped in the forestomach mucosa of rats given 200 mg EtAc/kg. Daily gavage administration of 100 mg/kg or 200 mg/kg EtAc to rats also caused microscopic lesions in the forestomachs (Table 1). In the 100 mg/kg EtAc-treated group, histo-
mucosal surface of formalin-fixed stomachs from rats given EtAc by Control; (b) 100 mg/kg, showing slight enlargement of the stomach; (c) thickening and folding of the forestomach; (d) 200 mg/kg showing the shafts embedded in the forestomach.
ETHYL
ACRYLATE-INDUCED
GASTRIC
TABLE INCIDENCE
Lesion Mucosal ulceration Mucosal hyperplasia Mucosal inflammation Hyperkeratosis Submucosal intlammation Foreign body reaction Submucosal fibrosis ’ Ethyl daily dose in groups b Time
1
OF HISTOPATHOLOGIC LESIONS IN THE FORESTOMACH OF RATS GIVEN AND KILLED AT VARYING TIMES AFTER THE LAST IXXE EtAc Control O/24 O/24 O/24 O/24 O/24 O/24 O/24
lb o/12 12112 o/12 12112 o/12 o/12 o/12
579
TOXICITY
EtAc’
EtAc
100 mg/kg I
O/8 618 O/8 618 018 O/8 O/8
FOR 14 DAYS
200 mg/kg
14
lb
14
018 018 018 018 018 018 I/8
8/12 12112 l/l2 12112 12112 o/12 o/12
O/8 818 3/g
w3 3/g
l/8 l/8
28 O/8
8/8 O/8 O/8 018 w3 318
acrylate was dissolved in corn oil to standardize dose volume at 5 ml/kg body weight, and the appropriate was given by gavage for 14 consecutive days. Control rats received 5 ml/kg corn oil alone and were killed with the corresponding EtAc-treated rats. (days) of termination after the last of 14 consecutive daily doses of EtAc.
pathologic changes in the forestomach consisted of marked generalized hyperkeratosis and moderate to marked generalized hyperplasia of the stratified squamous cell mucosal layer (Fig. 3 and Table 1). These and additional lesions were observed in the forestomach of rats which were given 200 mg/kg EtAc. Eight of twelve rats treated with 200 mg/kg EtAc had multiple mucosal ulcers with attendant purulent inflammation in adjacent mucosal epithelial layers. While there was marked generalized submucosal inflammation, this change was most severe in the vicinity of the mucosal ulcers (Fig. 3). The submucosal inflammation was characterized by infiltraton of neutrophils and lymphocytes and by early fibrosis (Fig. 3). All intact mucosal surfaces were hyperplastic and were covered by a thick layer of keratin (Fig. 3). One week after the last of 14 consecutive daily doses of 100 mg/kg EtAc, there was a decline in the incidence of the mucosal hyperplasia and hyperkeratosis of the forestomsch. These lesions completely disappeared by 2 weeks after the last dose (Table 1). Submucosal fibrosis was observed in one of eight rats 2 weeks after the last of 14 EtAc doses ( 100 mg/kg). In rats treated with 200 mg/kg EtAc for 14 consecutive days, there was a
complete disappearance of forestomach mucosal ulceration and a significant decline in the incidences of mucosal and submucosal inflammation within 2 weeks after the last dose (Table 1). Acute generalized inflammation (both mucosal and submucosal) disappeared within 4 weeks after the last 200-mg/kg EtAc dose. In contrast, the incidence of mucosal hyperplasia of the forestomach was unchanged 4 weeks after the last 200-mg/kg EtAc dose (Table 1). Hyperkeratosis was not observed in any animals at the end of this same recovery period. Two additional histologic changes were observed at increased incidence in the forestomachs of high dose rats at the end of the 4-week recovery period. Fibrosis, characterized by deposition of mature collagen, caused a thickening of the submucosa in three of eight rats given 200 mg/kg EtAc (Table 1 and Fig. 4). The second histologic change was multifocal foreign body reaction to embedded hair shafts and was present in the submucosa of all eight rats that had received 200 mg/kg EtAc (Table 1 and Fig. 5). The mucosa overlying these foreign body reactions was more severely hyperplastic than the adjacent mucosa. Similar changes were observed in only one rat following a 2-week recovery period.
GHANAYEM.
MARONPOT,
AND
MATTHEWS
a
of forestomachs from rats given EtAc by gavage for 14 consecutive days. (a) FIG. 3. Photomicrographs Control; (b) 100 mg/kg, showing hyperplasia and hyperkeratosis of the mucosal epithelium; (c) 200 mg/kg, showing mucosal ulceration (upper left), papillomatous hyperplasia of the mucosa (upper right), and severe submucosal inflammation. Hematoxylin and eosin stain. X I 15 (a and b) and X40 (c).
DISCUSSION Chronic gavage administration of 100 or 200 mg/kg EtAc to F344 rats (5 days/week for 2 years) caused hyperkeratosis, hyperplasia, squamous cell papillomas, and squamous cell carcinomas of the forestomach (NTP, 1983).
In subsequent studies (Ghanayem et al., 1985a), we demonstrated that gavage administration of similar doses of EtAc to F344 male rats caused acute lesions in both the forestomach and the glandular stomach. The present studies were conducted to expand upon our previous studies by characterizing gastric le-
FIG. 4. Submucosal layer thickened by mature fibrous connective tissue of rats treated with 14 consecutive daily doses of 200 mg/kg EtAc and killed 4 weeks after the last EtAc dose. Overlying epithelium hyperplastic. Hematoxylin and eosin. X64.
FIG. 5. A foreign body inflammatory reaction surrounds a hair shaft (arrow) embedded of rats treated with 14 consecutive daily doses of 200 mg/kg EtAc and killed 4 weeks dose. The overlying epithelium is hyperplastic. Hematoxylin and eosin. X53. 581
in the submucosa after the last EtAc
582
GHANAYEM.
MARONPOT.
sions induced by repetitive exposure to EtAc and by investigating the reversibility of EtAcinduced gastric lesions. Evidence of gastric toxicity was present in the forestomach of all rats treated with 14 consecutive daily gavage dosesof EtAc. However, EtAc-induced glandular stomach lesionsobserved after up to four consecutive daily dosesof EtAc (Ghanayem et al., 1985a) were not present following 14 consecutive daily gavage doses.Therefore, it appearsthat the glandular stomach recovered from acute effects and adapted to the effects of repeat exposure to EtAc with no persistent morphologic alterations. The forestomach, which was more sensitive to both the acute (Ghanayem et al., 1985a,b) and chronic effects (NTP, 1983) of EtAc, was also shown in the current studies to be more sensitive than the glandular stomach to repeated EtAc dosing ( 14 daily doses).Mucosal hyperplasia and hyperkeratosis of the forestomach were observed in all rats treated with 14 daily dosesof 100 or 200 mg/kg EtAc. In addition, mucosal ulceration as well as mucosal and submucosal inflammation were observed only in high-dose rats (200 mg/kg). Forestomach lesions in rats receiving EtAc were more numerous and more severe in the high-dose animals. This may explain the subsequent reversibility of EtAc-induced forestomach lesionsduring the recovery period in the low doserats versusthe persistenceof some of the alterations in the high dose rats (Table 1). Mucosal hyperplasia, which disappearedin animals receiving the low dose of EtAc, was lessseverebut still present in all animals killed 4 weeks following the last 200-mg/kg dose (Table 1). However, two lesions, submucosal fibrosis and foreign body reaction, became more prevalent in animals receiving the high doseas the recovery period progressed(Table I). The foreign body reaction and submucosal fibrosis represent lesions which were not observed at the end of the dosing regimen and must have developed during the recovery period. Furthermore, the foreign body reactions were apparently due to broken hair shaftsand/
AND
MATTHEWS
or feed particles entrapped in the forestomach mucosa during the healing process. What role, if any, the occurrence of foreign bodies(hair shaftsand/or feed particles) in the forestomachsof ethyl acrylate-treated rats may have in the subsequentdevelopment of benign and malignant forestomach tumors is unknown. However, experimental foreign body carcinogenesisis well establishedin the urinary bladder (Clayson and Pringle, 1966; Clayson et al., 1967) and subcutis (Farber and Cameron, 1980) of rodents, and potential counterparts in human cancer development include gallbladder tumors secondary to cholesterol stones, urinary bladder tumors secondary to chronic schistosomiasis,and lung tumors associated with asbestos(Farber and Cameron, 1980). It is conceivable that the foreign bodies observedin the present study might contribute to the forestomach tumor burden observed in the 2-year carcinogenesisstudy (NTP, 1983). We have demonstrated here that repetitive dosing with EtAc induces persistent proliferative changesin the forestomach of rats. It has been statedthat “induction of abnormally high levels of cell proliferation in a tissue provides a favorable environment for the development of cancer” (Iverson et ul., 1985). Experimental evidence that cellular proliferation secondary to injury is associatedwith enhanced susceptibility to chemical carcinogenesisat the site of cell proliferation hasbeen demonstrated for the glandular stomach (Nagai et al., 1984) as well as for the skin (Argyris and Slaga, 1981: Argyris, 1980). Similar observations have been made for liver (Pound and McGuire, 1978), urinary bladder (Clayson and Pringle, 1966) and mammary gland (Nandi, 1978). It is tempting to speculate that this phenomenon may be operational in the forestomach of rats given repeated gavage dosesof ethyl acrylate. Entrapment of foreign bodies and resultant reaction in the course of forestomach healing may serve to further exacerbate EtAc toxicity and cell proliferation. Further, EtAc is mutagenic in some systems(NTP, 1983) and increasedcell proliferation in its presencewould increasethe amount of replicating DNA at risk
ETHYL
ACRYLATE-INDUCED
for mutagenic alteration. Therefore, one or more of these factors alone or in combination may account for EtAc carcinogenicity to rat forestomach. Tissue changes, cell proliferation, foreign body reactions, and carcinogenicity are much more pronounced at EtAc doses of 200 mg/kg than at 100 mg/kg. Therefore, it may be that doses which produce increased and persistent cell proliferation and foreign body reactions would also be carcinogenic. REFERENCES ARGYRIS. T. S. (1980). Tumor promotion by abrasion induced epidermal hyperplasia in the skin of mice. J. Inwt. Dermatol. 75, 360-362. ARGYRIS. T. S., AND SLACA, T. J. (1981). Promotion of carcinomas by repeated abrasion in initiated skin of mice. Cancer ReJ. 41, 5193-5195. CLAYSON. D. B., AND PRINGLE, J. A. S. (1966). The influence of a foreign body on the induction of tumours in the bladder epithelium of the mouse. Brit. J. Cancer 20, 564-568. CLAYSON, D. B., PRINGLE. J. A. S.. AND BONSER.G. M. ( 1967). 4-Ethylsulphonylnaphthalene- I-sulphonamide: A new chemical for the study of bladder cancer in the mouse. Biochem. Pharmacol. 16, 619-626. FARBER, E., AND CAMERON, R. (1980). The sequential analysis of cancer development. In Advances in Cancer Research (F. C. Austin and C. W. Boone, eds.), Vol 3 I, pp. 125-226. Academic Press, New York. GHANAYEM, G. I., MARONPOT, R. R.. AND MATTHEWS, H. B. (1985a). Ethyl acrylate-induced gastric toxicity. I. Effect of single and repetitive dosing. Toxicol. Appl. Pharmacol. 80, 323-335. GHANAYEM, B. 1. MARONPOT. R. R., AND MATTHEWS, H. B. (1985b). Ethyl acrylate-induced gastric toxicity. II. Structure toxicity relationships and mechanism. To.uicol. .4pp,1.Pharmacol. 80, 336-344.
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TOBE. T. ( 1984). Susceptibility of healed gastric ulcers to chemical carcinogenesis in rats and implications of cellular kinetic changes. Canc~erRex 44, 5828-5835. NANDI, S. (1978). Hormonal carcinogenesis: A novel hypothesis for the role of hormones. J. Environ. Pathol. Toricol. 2, 13-20. National Toxicology Program (NTP) (1983). Carcino~qw esis Bioassa~~(!I Ethyl, lcrylate. ‘Technical Report Series 259, Publication (NIH) 82-25 15. U. S. Department of Health and Human Services. Public Health Service, National Institutes of Health. Research Triangle Park N. C. POUND. A. W.. AND MCGUIRE, L. J. (I 978). Influence of repeated liver regeneration on hepatic carcinogenesis by diethylnitrosamine in mice. Bri/. J. Cancer 37, 595602. U.S. CFR (1978). L:.S. Code qf.b>deral Regulatrons 21. 175-210. 176-180. 181-230. AND