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Induction of aberrant crypts in murine colon with varying sensitivity to colon carcinogenesis
LE’ITERS Cancer Letters 92 (1995) 209-214
Induction of aberrant crypts in murine colon with varying sensitivity to colon carcinogenesis Daniel W. Rosenberg*, Ying Liu Deportment of Phormoceutical Sciences, School of Pharmacy, University of Connecticut, Storm, CT 06269-2092, I:SA Received 10 March 1995; accepted 6 April 1995 -.-
.-.. ---~.
Abstract Repetitive treatmentwith the organotropic colon carcinogen,1,24hnethylhydrazine(DMH), producestumors in susceptible mousestrainsthat exhibit pathologicalfeaturesassociatedwith the human disease. As in humanpopulations, the geneticbackgroundof laboratory animalscomprisesa significantcomponentto this organ-specificcarcinogenesis, and several mouse lines, including AKR/J and DBAQJ are highly resistant to the tumorigenk e&%s of DMH. During the course of ongoing studies to establish phenotypic difTerencesbetween susceptibk (§WR/.l and P/J) and resistantstrains,we have examinedthe colonic mucosaof DMH-treated micefor the presenceof aberrantcrypt foci (ACF).*ACF representan early morphologicallesion in stepwise progression of colon cancer. In Experiment 1, 6-week-oldSWRNand AKRN micewereinjectedwith DMH (35 and 20mg!kg,respectively)oncea weekfor 2 weeks. Five weekslater, colons were removed and ACF visualized at low magnificationby light microscopyafter metbykme blue-staining.Only SWR/Jmicerevealedfocal atypia indicative of preneoplasticchange.To obtain additionalinformation about their morphology,tissuesectionscontainingACF were sectionedand stainedwith H&E. ACF are larger andhavea thicker epitheliallining than normalcrypts. H&E confirmedthe absence of theselesionsin untreatedSWIVJ and DMHexposed AKR/J mice.In Experiment2, SWRNand DBA/2J micewereinjectedwith DMH (35m&&g)once a weekfor 2 weeks.Nine weekslater, colonswereanalyzedfor ACF formation. Comparableto the first experiment, no ACF wereobservedin the colonic mucosaof the resistantDBAQJ line. In contrast, ACF werereadily identified in the middleand distal colons of similarly exposedSWRN mice. This differential responsebetweenresistantand susceptiblemouselinesfurther supportsan important role for ACF in the stepwiseprogressionof colon cancer. Keywords: Colon carcinogenesis; Mouse colon; 1,2-Dimethylhydrazine;Aberrant crypt -.._-- .. --
1. It&mdl&on
Repetitive treatment with the organotropic colon carcinogen, 1,2dimethylhydrazine (DMH), produces colon tumors in rodents exhibiting Abbreviations: DMH, 1,2dimethylhydrazine; H&E, hematoxylin and eosin; ACF, aberrant crypt foci. * Corresponding author. Elsevier Science Ireland Ltd. SSDI 0304-3835(95)03797-Z
.-.-.
pathological features that are similar to sporadic forms of human colon cancer [ 1,2]. As in human populations, the genetic background of laboratory animals comprises a significant component to this organ-specific carcinogenic response. Several laboratories have reported a marked intraspecific variability in colorectal tumorigenesis in mice exposed to DMH
[3-71. A tumor susceptibility
phenotype among mouse lines is thus, characteriz-
210
D. W. Rosenberg,
Y. Liu / Cancer
ed by tumor incidence (frequency and severity of tumors) in susceptible strains (SWR/J and P/J) that approaches 100%. In contrast, AKR/J and DBA/2J are largely resistant to the carcinogenic effects of DMH. In at least one strain, however, resistance can be partially overcome if carcinogen treatment is initiated at an early age [7]. Straindependency with respect to frequency and severity of tumors is summarized in Table 1. A number of diet-derived carcinogens, including benzo[a]pyrene, 2-amino-3,4-dimethyl-imidazo(4,5+quinoline (MeIQ), 5-hydroxy-2-furaldehyde and aflatoxin can produce neoplastic changes in the colons of rodents [9-131. The use of DMH, however, has overshadowed the use of other classes of chemical carcinogens. The DMH model, however, is not without limitations. While an adenoma to carcinoma progression has been clearly established in human colon cancer, adenomatous polyps are rarely observed in the DMH model and many tumors arise de novo from flat mucosa [ 141. Furthermore, the distribution and number of colon tumors is species-dependent. In rats, several large neoplasms are typically produced in the midcolon, while in mice, smaller and more numerous neoplasms develop, their distribution markedly skewed towards the distal colon [7,15,16]. Despite these qualifications, DMH has provided cancer researchers with the most reproducible experimental model for studying this disease. Aberrant crypt foci (ACF) are readily visible morphological changes within the colonic mucosa that may represent a critical event in the stepwise progression of colon cancer [8,17-201. ACF have been detected in rodent colon as early as 2 weeks
Table I Susceptibility of inbred mouse strains to DMH-induced cancer Resistant
Moderately susceptible
Highly susceptible
DBA/2J AKR/J C57BL/Ha
C57BL/6J BALBkHeA
P/J SWRIJ CFI ICRlHa STSIA
colon
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after carcinogen exposure [ 191. Sequential analyses of their growth and morphological characteristics suggest that these potentially premalignant lesions increase in size and number (multiplicity) over time and may exhibit dysplasia [8,17-201. Human ACF share morphological characteristics with those present in the rodent large bowel. Similar altered foci have been demonstrated in whole tissue mounts in grossly normal-appearing colon tissue resected from patients with colon cancer [21]. The predictive value of ACF as biomarkers for colon cancer risk, however, has recently been challenged. It is argued that the number and location of gut-associated lymphoid follicles, by acting via promotional mechanisms that have yet to be defined, are more reliably predictive of colonic mucosa at risk for neoplastic transformation [22,23]. During the course of ongoing studies to establih phenotypic differences between susceptible and resistant mouse lines [24], we have examined the colonic mucosa of DMH-treated mice for the presence of ACF. These lesions are readily identified in methylene blue-stained whole mount preparations of colon by alterations in the size and shape of the lumen, the epithelial cell lining and the pericryptal zone [8,17-201. We report that intraspecific differences in the extent of ACF formation correlate with reported tumor susceptibility to DMH, providing further evidence that the formation of ACF within the colonic mucosa are predictive for genetic susceptibility to chemicallyinduced colon cancer. 2. Materials and methods Five-six-week-old male SWR/J, AKRIJ and DBA/2J mice were purchased from The Jackson Laboratories (Bar Harbor, ME). All mice were housed four/cage in a temperature controlled, light-cycled (12 h light/dark) room. Mice had access to Purina Rodent Laboratory Chow (5001) and water ad libitum. DMH. 2HCl (Sigma Chemical Co., St. Louis, MO) was dissolved in 0.9% NaCl(l.5 mg EDTA/lOO ml) and pH adjusted to 6.5 with 1 M NaHCOs. Mice were injected S.C. with either 35 or 20 mg/kg DMH.2HCl or vehicle alone once per week over a 2-week period. Mice
D. W. Rosenberg, Y. Liu/Cancer
were eutha&ed by COZ asphyxiation at either 5 (E-t 1) or 9 (Experiment 2) weeks after the second injection. Immedi&ely after sacrifice, individual segme& of colon (proximal, middle and distd) were removed and fiushed free of fecal contents with PBS. Colons were then opened from cecum to anus and fixed flat in loo/o buffered formalin. After 24 h in fixative, colons were maintained for 15 min in 0.2% methylene blue and placed mucosal side up on a microscope slide to visualize crypt outlines. After visualization and photodocumentation, dye was removed and sections were prepared for analysis and confirmation of ACF by H&E. 3.Rcr#sddiseaaion In our first experiment, 6-week-old SWR/J and AIUUJ mice were treated S.C. with DMH (35 and 20 n@g b&y wt., respectively) once a week over a 2-w& period for a total of two doses. The dose! adn&istered to the AKR/J mice! was somewhat lower, due to the exter&e morbidity observed in this mouse line at higher doses (unpublished observations). Five weeks after the second injection, wIom were andymd for the presence of ACF. Altered foci were v&&ed at low magnification by light &roaqy ti methylene blue-staining. As shown in Fig. 1, a whole tissue mount of a methylene blue-stained colon from a DMH-treated SWWJ m revatkd a focal atypia (indicated by the arrow, panel a). To obtain additional information about the mqh&gy of ACF and to confirm their geographical position within the colonic epithelium, sactions containing ACF were marked and anbsddad in pe&& sectioned and stained with I-ME (Fig. 1, pan& b-f). The overaB d&rib&m of ACF in SWRIJ and AKIUJ wkms is in Table 2, Experiepithelii from an unmew l.!iaZ&n tread wool rmnme is shown in Fig. 1, panel b. This section &so e@$ains a lymph&d follicle, a colorric w t#& as described earlier [22,23] may be pr-cmhd t?fkcts OR tumor progressioa’pl]. No ACF m found in SWRIJ controls. , however, ACF were and distal colon of SWRIJ mk (Table 2). ACF observed are larger
Letters 92 11995) 209-214
and have a thicker epithelial lining than normalappearing crypts. The morphology of an early ACF produced in the distal colon of an SWR/J mouse is shown in Fig. 1, panel c. ACF were also found in two AKRIJ-treated mice, but they were only seen with methykae blue&&& arid not upon further examin&ion with H&B (Table 2). A representative se&ion from the c&m of carcinogen-exposed AKR/J is shown in Fq. 1 (panel d). There was m e&hoe of ACF fw in any of the whn ta emmined. b 8dditkm, there was no evidence of pathology produced by DMH in the liver, kidney or small intestie (data not shown). The absence of ACF in AKR/J mice is interesting in light of the inherent resistance of this mouse line to the carcino@c properties of DMH [4,5]. It is possible, however, that higher doses and/or longer exposure to carcinoga may ultimately elicit early morpho@&al changes associatetd with neopla& even in resia&mt Iines. A dose-response reiationship in female SpragueDawley rats has est&!i&ed a ptaaccOu elect on ACF formation at 100 m&g DMH (17). However, the dose of N that can be administered to AKR/J may be limited, as doses in excess of 30 mg&g have been found to produce severe morbidity (unpublished observation). Thus, it may not be possible, at least asing DMH, to overcome inkFeat re&ance UK&U&RID that protect the colonic epitheIium from cancerous progression. In Experiment 2, the effects of DMH on ACF formation in an 8dditiod tumor resistant mouse line, DBALZJ were tested. Fi -th-old SWWJ and DBALU mice were treated I.C. with DMH (35 mg&g) once a week over a 2-w& period. Nine weeks after the last inject& ti P penroved and ACF visu&&.ed a&r m&hb&+.taiuiug. The number of ACF and tl~&& titio~~w&in the colon a$#! qua&t#c!d in -r* 2 @xp&&mt 2). ComprtrabIe to the fuet tsqdaw~t, mi ACF war evident w&in the odanic msam of ~control aaisw6 h--r, colon sqgrmm& dSWR/J r&i @& 2). I% morphology of several ACF are shaam in Fig. 1 (panels e and f’). The lesions produced 10 weeks
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L-errers
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213
Table 2 Number and location of aberrant crypt foci and lymphoid follicles in DMH-treated mice -~..~ Strain
DMH
Experiment I ARRN + SWR/J + Experiment 2 DBAQJ + SWRIJ +
Proximal
Middle
Distal
0 0 0 0
0 0.60 * 0.40 0 2.40 zt 1.29
0 0.20 f 0.20 0 0.80 f 0.58
0 0 0.2 f 0.2 0.5 f 0.5
0 0 0 2.0
0 0 0 6.3 rt 2.8
l
1.1
------
Values represent the means f S.E.M. of a minimum of three to four mice per group
after treatment appeared different than those observed at 5 weeks (Experiment 1, panel c). There was more evidence of dysplasia in the component cells and a thicker epithelial lining was present. ACF were absent from colonic tissue isolated from similarly exposed tumor resistant DBA/2J mice (Table 2). These findings of strain-specific formation of ACF in a panel of inbred mouse lines treated with the methylating carcinogen, DMH, provide further evidence for the usefulness of this early morphological lesion as a biomarker for genetic cancer risk. The absence of ACF in two genetically resistant mouse lines (AKWJ and DBA/2J) suggest that protective factors are already operative prior to the appearance of altered colonic morphology. This sequence contrasts with the earlier findings of James et al. [7’j, who reported foci of dysplastic crypts throughout the large bowel of tumor resistant (C57BU6Ha) mice. Focal areas of atypism (FAA) were also reported in DMH-treated AKR/J
mice 1 week after the last of six injections [25]. Further studies are currently underway with dditional mouse lines with distinctly difTemnt tumor susceptibility patterns to establish the temporal sequence of morphologioal changes that occur within the colonic mucosa after carcinogen exposure. Using mouse lines at di&ring genetic risk for chemically-induced neoplastic transformation, a long-term goal of these studies is thus, to develop a greater understanding of the role of ACF in colon cancer progression.
These studies were supported in part by American Cancer Society Grant IN1 521-l 19 and by a generous gift from the Sandoz Pharmaceutical Research Institute (Hanover, NJ), The authors would like to thank Dr. Damon Averill for his many helpful suggestions.
Fig. I. Aberrant crypt foci in mouse colon. (a) Topographic view of unsectioned mouse colon from a DMH-treated SWR/J mouse stained with methykne blue (x 100). Arrow indicates a focus of three aberrant crypts surrounded by normal crypts. (b) Histological appearance of a lymphoid follicle (LF) from the colon of an DMH-treated SWR/J mouse. All crypts surrounding the Jymphoid tissue are normal appearing ( x 640). (c) Histological appearance of an early aberrant crypt surrounded by normal crypts from a colon 01 a DMH-treated SWR/J mouse, stained with H&E (arrow). (d) Histological appearance of the colon from a DMH-treated AKR/J mouse, stained with H&E. All crypts with circular openings are of a similar size and without dysplasia (X 640). (e,t) Cross-sections of two aberrant crypts surrounded by normal crypts from the distal colon of a DMH-treated SWR/J mouse stained with H&E. The two aberrant crypts are larger than the normal crypts with dysplasia ( x 640).
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References VI Druckrey, E. (1972) Organ-specific carcinogenesis in the digestive tract. In: Topics in Chemical Carcinogenesis, pp. 73-101. Editors: W. Nakahara, S. Takayama, T. Sugimura and S. Odashima. University Park Press, Baltimore, Maryland. PI Laqueur, G.L., Mickelson, 0. and Whiting, M.G. (1963) Carcinogenic properties of nuts from Cycas circinalis L. indigenous to Guam. J. Natl. Cancer Inst., 31, 919-951. I31 Diwan, B.A., Meier, H. and Blackman, K.E. (1977) Genetic differences in the induction of colorectal tumors by 1,2-dimethylhydtzine in inbred mice. J. Natl. Cancer Inst., 59, 455-458. [41 Evans, J.T., Hauschka, T.S. and Mittelman, A. (1974) Differential susceptibility of four mouse strains to induction of multiple large-bowel neoplasms by 1,2dimethylhydrazine. J. Natl. Cancer Inst., 52, 999-1000. PI Diwan B.A. and Blackman, K.E. (1980) Differential susceptibility of 3 sublines of C57BU6 mice to the induction of colorectal tumors by 1,2-dimethylhydraxine. Cancer Len., 9, 111-115. WI Evans, J.T., Shows, T.B., ‘Sproul, E.E., Paolini, N.S., Mittehnan, A. and Hauschka, T.S. (1977) Genetics of colon carcinogenesis in mice treated with 1,2-dimethylhydraxine. Cancer Res., 37, 134-136. 171 James, J.T., Shamsuddin, A.M. and Trump, B.F. (1983) Comparative study of the morphological, histochemical and proliferative changes induced in the large intestine of ICR/Ha and C57BVHa mice by 1,2-dimethylhydrazine. J. Natl. Cancer Inst., 71, 955-964. 181Bird, R.P. (1987) Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett. 37: 147-151. 191 Narasawa, T., Sato, T. and Hayakawa, M. (1971) Carcinoma of the colon and rectum of rats by rectal infusion of N-methyl-N’-nitro-N-nitrosoguanidine. Gann, 62, 231-234.
1101 Newbeme, P.M. and Rogers, A. (1973) Rat colon carcinomas associated with aflatoxin and marginal vitamin A. J. Natl. Cancer Inst., 50, 439-448. IIll Felton, J.S. Vanderlaan, M., Watkins, B.E., Hwang, M. and Knii, M.G. (1988) Monoclonal antibodies for the immunoassay of mutagenic compounds produced by cooking food. Environ. Mol. Mutagen., 11, 33. WI Zhang, X, Chow, C., Stamp, D., Minkin, S., Archer, M.C. and Bruce, W.R. (1993) Inititation and promotion of colonic aberrant crypt foci in rats by 5-hydroxy-2furaldehyde in thermolyzed sucrose. Carcinogenesis, 14, 773-775.
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LaMont, J.T. and O’Gorman, T.A. (1978) Experimental colon cancer. Gastroenterology, 75, 1157-l 169. 1141 Shamsuddin, A.K.M. and Trump, B.F. (1981) Colon epithelium, II: in vivo studies of colon carcinogenesis: light microscopic, histochemical and ultrastructural studies of histogenesis of azoxymethane-induced colon carcinomas in Fischer 344 rats. J. Natl. Cancer Inst., 66, 389-401. 1151 Thumherr, N. Deschner, E.E., Stonehill, E.H. and Lipkin, M. (1973) Induction of adenoarcinomas of the colon in mice by weekly injections of 1,2-dimethylhydraxine. Cancer Res., 33, 940-945. 1161 Barthold, SW. and Jonas, A.M. (1977) Morphogenesis of early 1,2-dimethylhydraxine-induced lesions and latent period reduction of colon carcinogenesis in mice by variant of Citrobacter frezmdii. Cancer Res., 37, 4352-4360. [I71 McLellan, E.A., Medline, A. and Bird, R.P. (1991) Dose response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon. Carcinogenesis, 12, 2093-2098. WI McLellan and Bird, R.P. (1988) Aberrant crypts: potential preneoplastic lesions in the murine colon. Cancer Res., 48, 6187-6192. iI91 McLellan, E.A., Medline, A. and Bird, R.P. (1991) Sequential analyses of the growth and morphological characteristics of aberrant crypt foci: putative preneoplastic lesions. Cancer Res., 51, 5270-5274. m Tudek, B., Bird, R.P. and Bruce, W.R. (1989) Foci of aberrant crypts in the colons of mice and rats exposed to carcinogens associated with foods. Cancer Res., 49, 1236-1240. WI Pretlow, T.P., Barrow, B.J., Ashton, W.S., O’Riordan, M.A., Pretlow, T.G., Jurcisek, J.A. and Stellato, T.A. (1991) Aberrant crypts: putative preneoplastic foci in human colonic mucosa. Cancer Res., 51, 1564-1567. WI Carter, J.W., Lancaster, H.K., Hardman, W.E. and Cameron, I.L. (1994) Distribution of intestine-associated lymphoid tissue, aberrant crypt foci, and tumors in the large bowel of 1,2-dimethylhydrazine-treated mice. Cancer Res., 54, 4304-4307. 1231 Hardman, W.E. and Cameron, I.L. (1994) Colonic crypts located over lymphoid nodules of 1,2-dimethylhydrazinetreated rats are hyperplastic and at high risk of forming adenocarcinomas. Carcinogenesis, 15, 2353-2361. [241 Rosenberg, D.W. and Mankowski, DC. (1994) Induction of Cyp2e-I protein in mouse colon. Carcinogenesis
1131
15, 73-78. [251
Deschner, E.E., Long, F.C. and Hakissian, B.A. (1988) Susceptibility to 1,2-dimethylhydraxine-induced colonic tumors and epithelial cell proliferation characteristics of Fl, F2 and reciprocal backcrosses derived from SWR/J and AKR/J parental mouse strains. Cancer 61.478-482.
Induction of aberrant crypts in murine colon with varying sensitivity to colon carcinogenesis Daniel W. Rosenberg*, Ying Liu Deportment of Phormoceutical Sciences, School of Pharmacy, University of Connecticut, Storm, CT 06269-2092, I:SA Received 10 March 1995; accepted 6 April 1995 -.-
.-.. ---~.
Abstract Repetitive treatmentwith the organotropic colon carcinogen,1,24hnethylhydrazine(DMH), producestumors in susceptible mousestrainsthat exhibit pathologicalfeaturesassociatedwith the human disease. As in humanpopulations, the geneticbackgroundof laboratory animalscomprisesa significantcomponentto this organ-specificcarcinogenesis, and several mouse lines, including AKR/J and DBAQJ are highly resistant to the tumorigenk e&%s of DMH. During the course of ongoing studies to establish phenotypic difTerencesbetween susceptibk (§WR/.l and P/J) and resistantstrains,we have examinedthe colonic mucosaof DMH-treated micefor the presenceof aberrantcrypt foci (ACF).*ACF representan early morphologicallesion in stepwise progression of colon cancer. In Experiment 1, 6-week-oldSWRNand AKRN micewereinjectedwith DMH (35 and 20mg!kg,respectively)oncea weekfor 2 weeks. Five weekslater, colons were removed and ACF visualized at low magnificationby light microscopyafter metbykme blue-staining.Only SWR/Jmicerevealedfocal atypia indicative of preneoplasticchange.To obtain additionalinformation about their morphology,tissuesectionscontainingACF were sectionedand stainedwith H&E. ACF are larger andhavea thicker epitheliallining than normalcrypts. H&E confirmedthe absence of theselesionsin untreatedSWIVJ and DMHexposed AKR/J mice.In Experiment2, SWRNand DBA/2J micewereinjectedwith DMH (35m&&g)once a weekfor 2 weeks.Nine weekslater, colonswereanalyzedfor ACF formation. Comparableto the first experiment, no ACF wereobservedin the colonic mucosaof the resistantDBAQJ line. In contrast, ACF werereadily identified in the middleand distal colons of similarly exposedSWRN mice. This differential responsebetweenresistantand susceptiblemouselinesfurther supportsan important role for ACF in the stepwiseprogressionof colon cancer. Keywords: Colon carcinogenesis; Mouse colon; 1,2-Dimethylhydrazine;Aberrant crypt -.._-- .. --
1. It&mdl&on
Repetitive treatment with the organotropic colon carcinogen, 1,2dimethylhydrazine (DMH), produces colon tumors in rodents exhibiting Abbreviations: DMH, 1,2dimethylhydrazine; H&E, hematoxylin and eosin; ACF, aberrant crypt foci. * Corresponding author. Elsevier Science Ireland Ltd. SSDI 0304-3835(95)03797-Z
.-.-.
pathological features that are similar to sporadic forms of human colon cancer [ 1,2]. As in human populations, the genetic background of laboratory animals comprises a significant component to this organ-specific carcinogenic response. Several laboratories have reported a marked intraspecific variability in colorectal tumorigenesis in mice exposed to DMH
[3-71. A tumor susceptibility
phenotype among mouse lines is thus, characteriz-
210
D. W. Rosenberg,
Y. Liu / Cancer
ed by tumor incidence (frequency and severity of tumors) in susceptible strains (SWR/J and P/J) that approaches 100%. In contrast, AKR/J and DBA/2J are largely resistant to the carcinogenic effects of DMH. In at least one strain, however, resistance can be partially overcome if carcinogen treatment is initiated at an early age [7]. Straindependency with respect to frequency and severity of tumors is summarized in Table 1. A number of diet-derived carcinogens, including benzo[a]pyrene, 2-amino-3,4-dimethyl-imidazo(4,5+quinoline (MeIQ), 5-hydroxy-2-furaldehyde and aflatoxin can produce neoplastic changes in the colons of rodents [9-131. The use of DMH, however, has overshadowed the use of other classes of chemical carcinogens. The DMH model, however, is not without limitations. While an adenoma to carcinoma progression has been clearly established in human colon cancer, adenomatous polyps are rarely observed in the DMH model and many tumors arise de novo from flat mucosa [ 141. Furthermore, the distribution and number of colon tumors is species-dependent. In rats, several large neoplasms are typically produced in the midcolon, while in mice, smaller and more numerous neoplasms develop, their distribution markedly skewed towards the distal colon [7,15,16]. Despite these qualifications, DMH has provided cancer researchers with the most reproducible experimental model for studying this disease. Aberrant crypt foci (ACF) are readily visible morphological changes within the colonic mucosa that may represent a critical event in the stepwise progression of colon cancer [8,17-201. ACF have been detected in rodent colon as early as 2 weeks
Table I Susceptibility of inbred mouse strains to DMH-induced cancer Resistant
Moderately susceptible
Highly susceptible
DBA/2J AKR/J C57BL/Ha
C57BL/6J BALBkHeA
P/J SWRIJ CFI ICRlHa STSIA
colon
Letters
92 (1995)
209-214
after carcinogen exposure [ 191. Sequential analyses of their growth and morphological characteristics suggest that these potentially premalignant lesions increase in size and number (multiplicity) over time and may exhibit dysplasia [8,17-201. Human ACF share morphological characteristics with those present in the rodent large bowel. Similar altered foci have been demonstrated in whole tissue mounts in grossly normal-appearing colon tissue resected from patients with colon cancer [21]. The predictive value of ACF as biomarkers for colon cancer risk, however, has recently been challenged. It is argued that the number and location of gut-associated lymphoid follicles, by acting via promotional mechanisms that have yet to be defined, are more reliably predictive of colonic mucosa at risk for neoplastic transformation [22,23]. During the course of ongoing studies to establih phenotypic differences between susceptible and resistant mouse lines [24], we have examined the colonic mucosa of DMH-treated mice for the presence of ACF. These lesions are readily identified in methylene blue-stained whole mount preparations of colon by alterations in the size and shape of the lumen, the epithelial cell lining and the pericryptal zone [8,17-201. We report that intraspecific differences in the extent of ACF formation correlate with reported tumor susceptibility to DMH, providing further evidence that the formation of ACF within the colonic mucosa are predictive for genetic susceptibility to chemicallyinduced colon cancer. 2. Materials and methods Five-six-week-old male SWR/J, AKRIJ and DBA/2J mice were purchased from The Jackson Laboratories (Bar Harbor, ME). All mice were housed four/cage in a temperature controlled, light-cycled (12 h light/dark) room. Mice had access to Purina Rodent Laboratory Chow (5001) and water ad libitum. DMH. 2HCl (Sigma Chemical Co., St. Louis, MO) was dissolved in 0.9% NaCl(l.5 mg EDTA/lOO ml) and pH adjusted to 6.5 with 1 M NaHCOs. Mice were injected S.C. with either 35 or 20 mg/kg DMH.2HCl or vehicle alone once per week over a 2-week period. Mice
D. W. Rosenberg, Y. Liu/Cancer
were eutha&ed by COZ asphyxiation at either 5 (E-t 1) or 9 (Experiment 2) weeks after the second injection. Immedi&ely after sacrifice, individual segme& of colon (proximal, middle and distd) were removed and fiushed free of fecal contents with PBS. Colons were then opened from cecum to anus and fixed flat in loo/o buffered formalin. After 24 h in fixative, colons were maintained for 15 min in 0.2% methylene blue and placed mucosal side up on a microscope slide to visualize crypt outlines. After visualization and photodocumentation, dye was removed and sections were prepared for analysis and confirmation of ACF by H&E. 3.Rcr#sddiseaaion In our first experiment, 6-week-old SWR/J and AIUUJ mice were treated S.C. with DMH (35 and 20 n@g b&y wt., respectively) once a week over a 2-w& period for a total of two doses. The dose! adn&istered to the AKR/J mice! was somewhat lower, due to the exter&e morbidity observed in this mouse line at higher doses (unpublished observations). Five weeks after the second injection, wIom were andymd for the presence of ACF. Altered foci were v&&ed at low magnification by light &roaqy ti methylene blue-staining. As shown in Fig. 1, a whole tissue mount of a methylene blue-stained colon from a DMH-treated SWWJ m revatkd a focal atypia (indicated by the arrow, panel a). To obtain additional information about the mqh&gy of ACF and to confirm their geographical position within the colonic epithelium, sactions containing ACF were marked and anbsddad in pe&& sectioned and stained with I-ME (Fig. 1, pan& b-f). The overaB d&rib&m of ACF in SWRIJ and AKIUJ wkms is in Table 2, Experiepithelii from an unmew l.!iaZ&n tread wool rmnme is shown in Fig. 1, panel b. This section &so e@$ains a lymph&d follicle, a colorric w t#& as described earlier [22,23] may be pr-cmhd t?fkcts OR tumor progressioa’pl]. No ACF m found in SWRIJ controls. , however, ACF were and distal colon of SWRIJ mk (Table 2). ACF observed are larger
Letters 92 11995) 209-214
and have a thicker epithelial lining than normalappearing crypts. The morphology of an early ACF produced in the distal colon of an SWR/J mouse is shown in Fig. 1, panel c. ACF were also found in two AKRIJ-treated mice, but they were only seen with methykae blue&&& arid not upon further examin&ion with H&B (Table 2). A representative se&ion from the c&m of carcinogen-exposed AKR/J is shown in Fq. 1 (panel d). There was m e&hoe of ACF fw in any of the whn ta emmined. b 8dditkm, there was no evidence of pathology produced by DMH in the liver, kidney or small intestie (data not shown). The absence of ACF in AKR/J mice is interesting in light of the inherent resistance of this mouse line to the carcino@c properties of DMH [4,5]. It is possible, however, that higher doses and/or longer exposure to carcinoga may ultimately elicit early morpho@&al changes associatetd with neopla& even in resia&mt Iines. A dose-response reiationship in female SpragueDawley rats has est&!i&ed a ptaaccOu elect on ACF formation at 100 m&g DMH (17). However, the dose of N that can be administered to AKR/J may be limited, as doses in excess of 30 mg&g have been found to produce severe morbidity (unpublished observation). Thus, it may not be possible, at least asing DMH, to overcome inkFeat re&ance UK&U&RID that protect the colonic epitheIium from cancerous progression. In Experiment 2, the effects of DMH on ACF formation in an 8dditiod tumor resistant mouse line, DBALZJ were tested. Fi -th-old SWWJ and DBALU mice were treated I.C. with DMH (35 mg&g) once a week over a 2-w& period. Nine weeks after the last inject& ti P penroved and ACF visu&&.ed a&r m&hb&+.taiuiug. The number of ACF and tl~&& titio~~w&in the colon a$#! qua&t#c!d in -r* 2 @xp&&mt 2). ComprtrabIe to the fuet tsqdaw~t, mi ACF war evident w&in the odanic msam of ~control aaisw6 h--r, colon sqgrmm& dSWR/J r&i @& 2). I% morphology of several ACF are shaam in Fig. 1 (panels e and f’). The lesions produced 10 weeks
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Table 2 Number and location of aberrant crypt foci and lymphoid follicles in DMH-treated mice -~..~ Strain
DMH
Experiment I ARRN + SWR/J + Experiment 2 DBAQJ + SWRIJ +
Proximal
Middle
Distal
0 0 0 0
0 0.60 * 0.40 0 2.40 zt 1.29
0 0.20 f 0.20 0 0.80 f 0.58
0 0 0.2 f 0.2 0.5 f 0.5
0 0 0 2.0
0 0 0 6.3 rt 2.8
l
1.1
------
Values represent the means f S.E.M. of a minimum of three to four mice per group
after treatment appeared different than those observed at 5 weeks (Experiment 1, panel c). There was more evidence of dysplasia in the component cells and a thicker epithelial lining was present. ACF were absent from colonic tissue isolated from similarly exposed tumor resistant DBA/2J mice (Table 2). These findings of strain-specific formation of ACF in a panel of inbred mouse lines treated with the methylating carcinogen, DMH, provide further evidence for the usefulness of this early morphological lesion as a biomarker for genetic cancer risk. The absence of ACF in two genetically resistant mouse lines (AKWJ and DBA/2J) suggest that protective factors are already operative prior to the appearance of altered colonic morphology. This sequence contrasts with the earlier findings of James et al. [7’j, who reported foci of dysplastic crypts throughout the large bowel of tumor resistant (C57BU6Ha) mice. Focal areas of atypism (FAA) were also reported in DMH-treated AKR/J
mice 1 week after the last of six injections [25]. Further studies are currently underway with dditional mouse lines with distinctly difTemnt tumor susceptibility patterns to establish the temporal sequence of morphologioal changes that occur within the colonic mucosa after carcinogen exposure. Using mouse lines at di&ring genetic risk for chemically-induced neoplastic transformation, a long-term goal of these studies is thus, to develop a greater understanding of the role of ACF in colon cancer progression.
These studies were supported in part by American Cancer Society Grant IN1 521-l 19 and by a generous gift from the Sandoz Pharmaceutical Research Institute (Hanover, NJ), The authors would like to thank Dr. Damon Averill for his many helpful suggestions.
Fig. I. Aberrant crypt foci in mouse colon. (a) Topographic view of unsectioned mouse colon from a DMH-treated SWR/J mouse stained with methykne blue (x 100). Arrow indicates a focus of three aberrant crypts surrounded by normal crypts. (b) Histological appearance of a lymphoid follicle (LF) from the colon of an DMH-treated SWR/J mouse. All crypts surrounding the Jymphoid tissue are normal appearing ( x 640). (c) Histological appearance of an early aberrant crypt surrounded by normal crypts from a colon 01 a DMH-treated SWR/J mouse, stained with H&E (arrow). (d) Histological appearance of the colon from a DMH-treated AKR/J mouse, stained with H&E. All crypts with circular openings are of a similar size and without dysplasia (X 640). (e,t) Cross-sections of two aberrant crypts surrounded by normal crypts from the distal colon of a DMH-treated SWR/J mouse stained with H&E. The two aberrant crypts are larger than the normal crypts with dysplasia ( x 640).
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