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Original article
Traditional serrated adenomas and serrated carcinomas in carcinogen-treated rats
  1. Carlos A Rubio
  1. Correspondence to Professor Carlos A Rubio, Gastrointestinal and Liver Pathology Research Laboratory, Department of Pathology, Karolinska Institute and University Hospital, Stockholm 17176, Sweden; Carlos.Rubio{at}ki.se

Abstract

Aims A recent review of archived sections from early experiments in rats showed neoplasias exhibiting serrated configurations. The aim was to assess the frequency of serrated neoplasias in the colon and small intestine of carcinogen-treated rats.

Methods While reviewing archival sections from early experiments in Sprague-Dawley (SD) and Fisher-344 (F-344) rats, we recently detected colonic and intestinal traditional serrated adenomas (displaying serrated or microtubular patterns) and serrated carcinomas. SD rats were injected 1,2-dimethylhydrazine (DMH) for 27 weeks whereas F-344 rats were fed with a pyrolysate (GLU-1) for 24 months. Filed sections from 358 colonic and small intestinal neoplasias were re-evaluated.

Results DMH-treated SD rats had 215 colonic neoplasias (1.4% were serrated adenomas, 7.9% microtubular adenomas, 2.8% serrated carcinomas and 2.8% microtubular carcinomas). GLU1-treated F-344 rats had 53 colonic neoplasias (1.9% were serrated adenomas and 20.8% microtubular adenomas), and 89 small intestinal neoplasias (1.1% were serrated adenomas, 42.7% microtubular adenomas and 6.7%, microtubular carcinomas).

Conclusions DMH/SD-rats develop serrated and microtubular adenomas and carcinomas in the colon, whereas GLU1/F-344 rats develop microtubular adenomas in the colon and microtubular adenomas and carcinomas in the small intestine. The two rat-settings emerge as suitable models to study the molecular attributes of serrated and microtubular neoplasias under the standard conditions of the laboratory. This study is the first showing that a substantial number of serrated and particularly microtubular adenomas and carcinomas develop in the colon and small intestine of experimental rats. Importantly, serrated and microtubular neoplasias in rats recreate the histology of duodenal and colonic traditional serrated neoplasias in human beings.

  • HISTOPATHOLOGY
  • COLORECTAL CANCER
  • COLON
  • CANCER RESEARCH

https://doi.org/10.1136/jclinpath-2016-204037

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    Introduction

    While attempting to produce amyotrophic lateral sclerosis with nuts of Cycas circinalis (a tropical fern from a family of Cycadaceae), Laqueur et al1 accidentally found that rats had developed colonic cancer. The same author subsequently demonstrated that the active carcinogen was cycasin, a water soluble á-glucoside of methylazoxymethanol.2 ,3 That discovery, lead Druckrey et al4 to administer a structurally similar compound, 1,2-dimethylhydrazine (DMH), to rats.

    DMH produce tumours, predominantly in the colon,5–8 and in the colon and small intestine.9–12 In later years a vast literature on colorectal neoplasias evoked by different carcinogens, by genetic engineering or by spontaneous mutations in mice and rats has been published. A recent survey on colonic tumours in rats and mice yielded 16 829 publications (PUBMED, 4 July 2016). This vast literature is a testimony of the expectations that experimental models might contribute to grasp the elusive process of colorectal carcinogenesis in human beings.

    DMH and its carcinogenic metabolites (azoxymethane (AOM) and methylazoxymethanol) are today, the most commonly used compounds to study morphology, pathogenesis, prevention and treatment of experimentally induced colonic tumours.12–15 Additives in the diet such as fat or fibres,16–18 and chemicals19 have also been used to study possible prevention of colonic tumours in rodents. Although the results have not been conclusive, the current understanding is that daily lifestyle, especially dietary habits, are important factors in the development of human cancer. In this context, early experiments demonstrated that extracts of scorched broiled fish and meat contained highly mutagenic heterocyclic amines.20 Accordingly, pyrrolate from scorched amino acids and proteins were given to rodents to study potential tumourogenesis. Masuda and Takayama et al21 found that the oral administration of 2-amino-6-methyldipyrido[1,2-a:3′,3′-d]imidazole (GLU-1) isolated from a glutamic acid pyrrolate, induced tumours in the small and large intestine, liver, ear duct and clitoral gland in F-344 rats. When the same pyrrolate was orally administered to CDF1 mice [(BALB/cAnN×DBA/2N)F1], no tumours were found in the small or the large intestine.21

    The aforementioned carcinogens usually generate adenomas and carcinomas. Adenomas have been histologically classified into tubular, villous and a mixed type (tubulovillous)13 ,22–24 and carcinomas into well differentiated (tubular or villous), poorly differentiated (mucinous, signet-ring cell), undifferentiated13 ,25–28 and gut-associated lymphoid tissue (GALT) carcinomas.12 ,29 In later years, serrated configurations and serrated adenomas have been induced in the small intestine of rodents by crossing Cyp1a1Cre-Apc (ApcHET) mice with Indian hedgehog (SHH)-overexpressing mice treated with β-naphthoflavone.30 Davies et al31 found serrated adenomas and invasive carcinomas in the small intestine using Pten and Kras-mutated Apcfl/+ mice. In the small intestine of wild-type Pten and Kras-mutated mice hyperplastic polyps, dysplastic sessile serrated adenomas and metastasising adenocarcinomas with serrated features unexpectedly occurred. Following activation of a KrasG12D mutant allele or inactivated Apc alleles Feng et al32 recorded hyperplastic and serrated configurations in the colon epithelium of mice. Bongers et al33 found that the transgenic expression of the epidermal growth factor receptor in conjunction with ligand heparin-binding epidermal growth factor (EB-EGF), promoted the development of small caecal serrated polyps in mice. None of these polyps were serrated adenomas. With a spontaneous risen splice site mutation in the murine Smad4 genes, Hohenstein et al34 elicited serrated adenomas and mixed polyposis in the upper digestive tract of mice. Timely, serrated adenomas in the upper digestive tract were recently found in human beings.35

    For many years, the general view was that the vast majority of the human colorectal carcinomas develop from conventional adenomas via the adenoma–carcinoma sequence.36 More recently serrated colorectal polyps (hyperplastic polyps, sessile serrated polyps and traditional serrated adenomas (TSAs37) have emerged as an alternative pathway of colorectal carcinogenesis.38 It has been estimated that about 30% of the colorectal cancer (CRC) in human beings progress via the serrated pathway.39

    To evoke colonic neoplasias in rodents we previously used two different carcinogens: DMH6 and GLU1.40 Lately, while reviewing archived sections from those early experiments6 ,19 ,22–26 ,29 ,40–42 we detected colonic neoplasias exhibiting serrated configurations. The purpose of the present communication was to review a cohort of archived sections from early experiments, to assess the frequency of serrated neoplasias in the colon. Tumours in the small intestine (not included in previous publications) were also reviewed.

    Methods

    Filed histological sections from 235 male Sprague-Dawley (SD) rats and from 101 Fisher-344 (F-344) rats, were reviewed.

    SD rats were injected subcutaneously with a weekly dose of 21 mg/kg body weight of DMH, hydrochloride salt Nw 133.02 suspended in 1 mL EDTA solution, for 27 weeks.

    F-344 rats were fed with pellets containing 500 ppm of the mutagenic pyrolysate compound 2-amino-6-methyldipyrido imidazole (Glu-P-1), for 24 months.

    Definitions

    Adenomas

    Grossly visible circumscribed lesions having crowded crypts lined with dysplastic cells. Adenomas were histologically classified into tubular, villous and TSA. TSA were subclassified into those with dysplastic open serrations (figures 1 and 5) as originally described by Longacre and Fenoglio-Preiser43 and those with dysplastic microtubules (figures 2 and 6), displaying dysplastic closed rings arranged within and/or lengthwise, elongated outgrowths, as previously described.44–47 The microtubular adenoma has also been referred to as with ectopic crypt formations (ECFs).37

    Figure 1
    Figure 1

    Serrated adenoma (colon, H&E. Upper panel: 1,2-dimethylhidrazine-treated Sprague-Dawley rats. Lower panel: F-344 GLU1-treated rats. Clockwise: ×10, ×20, ×40, ×20).

    Figure 2
    Figure 2

    Microtubular adenoma (colon, H&E. Upper panel: 1,2-dimethylhidrazine-treated Sprague-Dawley rats. Lower panel: F-344 GLU1-treated rats Clockwise: ×10, ×10, ×20, ×40). Note marked cytoplasmic eosinophilia (upper-right quadrant).

    Carcinomas

    Neoplastic glands invading through the muscularis mucosae into the submucosal layer or beyond. Histologically they were classified into high or low differentiated (mainly tubular), signet-ring cell carcinoma, undifferentiated carcinomas13 and GALT carcinomas.29 Following histological findings in human colonic carcinomas, carcinomas in rats were further subclassified into villous, serrated (figure 3) and microtubular.45–49 The descriptive term microtubular carcinoma44 was preferred to ‘ECF-carcinoma’, since it reflected the glandular configurations appearing in the former carcinomas48 (figures 4 and 7). In fact, all adenocarcinoma-phenotypes in the colon and small intestine are built of ectopic neoplastic glands, both in rats and in human beings.

    Figure 3
    Figure 3

    Serrated carcinoma (colon, H&E. Clockwise: ×4, ×10, ×20, ×10).

    Figure 4
    Figure 4

    Microtubular carcinoma (colon, H&E. Clockwise: ×2, ×4, ×20, ×10).

    Enteric tumours

    Tumours found in the small intestine.

    Statistical analysis

    The non-parametric Mann-Whitney U test was applied, to compare difference between groups. Statistical significance was defined as p<0.05.

    The Ethical Committee of the Karolinska Institute approved the old experiments in Sweden (N 48/1989).

    Results

    Tumours in the colon

    A total of 268 colonic neoplasias were found: 215 in 235 DMH-treated rats and 53 in 101 GLU1-treated rats (table 1). The comparative histological classification of the neoplasias found in the two groups of animals is shown in table 2.

    1. Colonic adenomas: the table shows that colonic adenomas occurred in 85.0% of all neoplasias in GLU1-treated rats, but only in 20.4% of all neoplasias in DMH-treated animals (p<0.05). Table 2 also shows that the proportion of serrated adenomas (figure 1) was similar in both DMH and GLU1 groups (1.4% and 1.9%, respectively). On the other hand, microtubular adenomas (figure 2) accounted for 20.8% of the colonic neoplasias in GLU1-treated rats, but only for 7.9% in DMH-treated rats (p<0.05). Tubular adenomas were more often found in GLU1-treated (60.4%) than in DMH-treated (10.2%), (p<0.05).

    2. Colonic carcinomas: in DMH-treated rats, 2.8% of the colonic neoplasias were serrated carcinomas (figure 3), 2.8% microtubular carcinomas (figure 4), 39.1% tubular carcinomas, 12.6%, signet-ring cell carcinomas and 20.5% GALT carcinomas.

    View this table:
    Table 1

    Histological phenotypes of neoplasias found in the colon of 215 SD rats treated with DMH, and in 53 F-344 rats treated with GLU1

    View this table:
    Table 2

    Histological phenotypes of neoplasias found in the small intestine of 89 F-344 rats treated with GLU1 and of one SD rat treated with DMH

    In the colon of GLU1-treated rats only tubular carcinomas were recorded: they accounted for 15.1% of all neoplasias in table 2. The difference between the proportions of tubular carcinomas in DMH-treated and in GLU1-treated rats was significant (p<0.05).

    Tumours in the small intestine

    A total of 90 intestinal neoplasias were found: 89 in GLU1-treated rats and one in DMH-treated rats.

    1. Intestinal adenomas: the comparative histological classification of the intestinal neoplasias in table 2 reveals that out of the 89 neoplasias in the small intestine of GLU1-treated rats, 1.1% serrated adenomas (figure 5), 42.7% were microtubular adenomas (figure 6), 17.9% tubular adenomas and 23.6% villous adenomas. In the small intestine of DMH-treated rats, no adenomas were found.

    2. Intestinal carcinomas: in GLU1-treated rats 6.7% of the intestinal neoplasias were microtubular carcinomas (figure 7) and 7.9%, tubular carcinomas. The only invasive neoplasia found in the small intestine of DMH-treated rats was of intestinal type.

    Figure 5
    Figure 5

    Serrated adenoma (small intestine, H&E. Upper panel: 1,2-dimethylhidrazine-treated Sprague-Dawley rats. Lower panel: F-344 GLU1-treated rats. Clockwise: ×20, ×20, ×20, ×20).

    Figure 6
    Figure 6

    Microtubular adenoma (small intestine, F-344 GLU1-treated rats, H&E. Clockwise: ×4, ×10, ×10, ×20, ×10, ×12).

    Figure 7
    Figure 7

    Microtubular carcinoma (small intestine, F-344 GLU1-treated rats, H&E. Clockwise: ×10, ×10, ×10, ×10).

    Discussion

    In many studies, the administration of DMH and AOM to rodents triggered neoplasias located mainly in the colon. In AOM-treated F-344 rats, Finley et al18 found colon carcinomas in 70%, Zalatnai et al50 in 83% and Guillem et al51 exclusively in the colon. In SD rats treated with DMH, Schmähl et al5 found colonic tumours in 72%, Gershbein and Rao10 mainly colonic carcinomas and Chen et al52 only colonic tumours. Tumours in the small intestine were not found. Nauss et al53 noticed mainly colonic tumours in both SD and F-344 rats treated with DMH on a 20% fat diet, and Balansky et al7 recorded mainly colonic tumours. In previous studies of SD rats treated with DMH we also documented mainly colonic6 ,19 ,22–26 ,41 ,54 and ear tumours.55 In a study of DMH-treated SD rats fed with a guar gum diet, Gershbein et al8 found mainly small intestinal carcinomas, Hirata et al11 tumours from the duodenum to the colon as well as exclusively in the colon in methylnitrosourea (MNU)-treated SD rats. In surgically manipulated DMH-treated SD rats Morgenstern et al56 found tumours mainly in the proximal duodenum. Why using the same strain of rats and the same carcinogen some authors found colonic tumours only, others colonic and enteric tumours and yet others mainly small intestinal carcinomas, is puzzling. In the present retrospective study we found significantly more frequent colonic adenomas in GLU1-treated rats, than in DMH-treated animals. Small intestinal tumours evolved almost exclusively in the small intestine of GLU1-treated rats. Notably, serrated adenomas, microtubular adenomas and microtubular carcinomas were exclusively found in the small intestine of GLU1-treated F-344 rats. The cause for the organotropic preference of GLU1 to generate serrated neoplasias in the small intestine remains a conundrum. Possible candidates for the organotropic predilection are: (i) the type of carcinogen used, since the chemical structures of GLU1 and DMH were completely different, (ii) the dose of carcinogen administered, since GLU1 was given, daily for 2 years, whereas DMH was administered once a week, for 27 weeks and (iii) the route of administration, since GLU1 was included in the diet (pellets) while DMH was injected subcutaneously. Less likely candidates for the organotropic preference of GLU1 are: (i) the strain of the rodent, since Takayama et al22 found no tumours in the small or the large intestine in GLU1-treated CDF1 mice [(BALB/cAnN×DBA/2N)F1], (ii) the gender of the animals, since only male F-344 and SD male rats were used in both experiments and (iii) the age of the rats: this possibility was also rejected since rats of the same age could display different neoplastic configurations (tubular, villous, serrated or microtubular). Hence, there was no indication that tubular or villous neoplasias had chronologically ‘re-modelled’ into serrated or microtubular neoplasias or that the latter two were transitional patterns capable of changing into tubular or villous neoplastic phenotypes with increasing age. Thus, the chemical structure of the carcinogen, the route of administration and/or the dose given to the animal might account for the differences in the topographical localisation of the neoplasias (enteric vs colonic). Another crucial question is: which are the molecular signals that trigger serrated and microtubular neoplastic configurations in F-344 and in SD rats? In a seminal work, Harris et al57 studied the molecular pathway of development of the initial formation of a meristic pattern of barb ridges in birds. These authors found that the variation in barb morphogenesis including the simple, tufted, plumulaceous natal down feather of a chick, the fusion of barb ridges creating the rachis ridge and the branched feather forms were controlled by the interaction between Sonic hedgehog and bone morphogenetic protein-2. Based on this knowledge we are prone to speculate that the accumulation of cellular mutations leading to neoplasia (ie, carcinogenesis) might act independently from the series of molecular signals that choreograph dissimilar structural configurations (ie, morphogenesis). Whether disparate carcinogens in the microenvironment are able to induce dissimilar mutations in colorectal stem cells58 resulting in different architectural neoplastic configurations48 remains to be explored.

    This study reveals that in addition to the classical histological classification of adenomas in rodents challenged with DMH13 and with GLU1,40 serrated and microtubular adenomas should be integrated. The failure to recognise the latter two prototypes in previous publications with this material is regrettable. One mitigating circumstance might be that the recent awareness of serrated neoplasias in the colon38 ,43 ,49 ,59 and in the upper digestive tract35 in human beings might have prompted their detection in rats.

    Microtubular adenomas were considerably more frequent in the colon of GLU1-treated rats, whereas microtubular and serrated carcinomas occurred exclusively in the colon of DMH-treated animals. The latter suggests that colonic microtubular and serrated adenomas in DMH-treated SD rats are aggressive neoplasms, prone to evolve into invasive carcinoma.

    This is the first study showing that a substantial number of serrated and particularly microtubular neoplasias develop in the colon of DMH-treated SD rats and in the colon and small intestine of GLU1-fed F-344 rats. Although the cause(s) for the different histological behaviour and their distinctive localisation (enteric vs colonic) in the two group of animals remains unsettled, it is not inconceivable that GLU1 binds, mutates, suppresses or interacts with oncogenes in intestinal and colonic epithelial cells, in a different fashion than DMH does.

    It is submitted that DMH-treated SD rats and GLU1-treated F-344 rats might be useful experimental models to induce enteric and colonic serrated and microtubular neoplasias and to explore their molecular attributes under the standard conditions of the laboratory. Importantly, serrated and microtubular neoplasias in the two rats paradigms recreate the histology of duodenal and colonic traditional serrated neoplasias reported in human beings.35 ,44 ,45 ,49 ,59

    Take home messages

    • Traditional serrated adenomas (TSAs) are part of the serrated pathway of colorectal carcinogenesis in human beings.

    • Two carcinogens: 1,2-dimethylhydrazine (DMH) and GLU1 were administered to Sprague-Dawley (SD) rats and to F-344 rats, respectively.

    • TSA were subclassified into those with open serrated crypts and those with closed microtubules.

    • DMH-treated SD-rats developed serrated and microtubular adenomas and serrated and microtubular carcinomas in the colon.

    • GLU1 treated F344 rats developed microtubular adenomas in the colon and microtubular adenomas and microtubular carcinomas in the small intestine.

    • Serrated and microtubular neoplasias in rats recreate the histology of duodenal and colonic traditional serrated neoplasias in human beings.

    • The two rat-settings emerge as suitable models to study the molecular attributes of serrated and microtubular neoplasias under the standard conditions of the laboratory.

    Acknowledgments

    The author is indebted to Dr Shozo Takayama, President of the Princess Takamatsu Cancer Research Fund, Tokyo, Japan, for permitting me to review, once again, his experiments on GLU1-treated F-344 rats.

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    Footnotes

    • Handling editor Cheok Soon Lee

    • Competing interests None declared.

    • Ethics approval The Ethical Committee of the Karolinska Institute approved the old experiments in Sweden (N 48/1989).

    • Provenance and peer review Not commissioned; externally peer reviewed.