Wound healing and colon carcinogenesis. Enhancing effects of skin wounding on development of colon tumors induced by 1,2 Dimethylhydrazine in the rat

Cicatrização e carcinogênese do colon: influência da cicatrização cutânea no modelo de desenvolvimento de tumores do colon induzido pela 1,2 dimetil-hidrazina no rato

Abstracts

Este estudo foi desenvolvido com a finalidade de investigar a possível influência do processo de cicatrização sobre o desenvolvimento neoplásico à distância, em um modelo experimental de carcinogênese do colon induzido pela 1,2 dimetil-hidrazina ( DMH). Ratos Wistar machos receberam injeções semanais de DMH ( 20mg/Kg, via subcutânea) ou solução salina, durante oito semanas. Na nona semana, um grupo tratado com DMH e um controle, foram submetidos à intervenção cirúrgica para retirada de um retalho cutâneo de 4cm no flanco direito, que cicatrizou por segunda intenção. Na 12ª semana, logo após o fechamento da ferida cutânea, os animais foram submetidos à eutanásia. O colon foi dividido em segmentos proximal e distal e examinado a nível macroscópico e histológico. Foram analisadas a incidência, distribuição e morfologia das lesões. O número total de tumores na mucosa do colon e o número de tumores por animal foi significantimente maior no grupo submetido à ferida cutânea do que no grupo tratado somente com DMH. O número de carcinomas pouco diferenciados foi significantimente maior no grupo com ferida cutânea do que em seu respectivo controle. Estes resultados sugerem que o processo de reparação de uma ferida cutânea favorece o desenvolvimento neoplásico em um órgão à distância, tal como o colon e que este efeito parece estar relacionado ao tipo histológico da neoplasia.

Cicatrização de feridas; Cólon; 1,2 dimetil-hidrazina; Ratos; Carcinógenos

WOUND HEALING AND COLON CARCINOGENESIS. ENHANCING EFFECTS OF SKIN WOUNDING ON DEVELOPMENT OF COLON TUMORS INDUCED BY 1,2 DIMETHYLHYDRAZINE IN THE RAT¹

Júlio Lopes Sequeira²

Shoiti Kobayasi³

Maria Aparecida Marchesan Rodrigues⁴

Sequeira JL, Kobayasi S, Rodrigues MAM. Wound healing and colon carcinogenesis: enhancing effects of skin wounding on development of colon tumors induced by 1,2 dimethylhydrazine in the rat. Acta Cir Bras [serial online] 2000 Jul-Sept;15(3). Available from: URL: http://www.scielo.br/acb.

ABSTRACT: This study demonstrates the tumor promoting effect at a distant site of skin wounding, in a model of colon carcinogenesis induced by 1,2 dimethylhydrazine (DMH) in the rat. Six-week-old male Wistar rats were given subcutaneous injections of DMH, 20mg/kg, or saline, once a week, for eight weeks. One week after the last DMH injection the animals received a full thickness skin wound in their dorsal skin and the wound was left open to heal by second intention. Control and DMH-treated rats, with or without skin wounds were killed at the 12th week, just after healing of the skin wound was complete. The colons were removed and divided into proximal and distal parts. Each segment was rolled as "Swiss roll"and processed for histology. The incidence, distribution and morphology of the colon tumors was recorded. The total number of tumors in the colonic mucosa and the number of tumors per rat was significantly higher in the skin-wounding DMH- treated group than in the unwounded group. In the histopathological analysis of the colon the number of poorly differentiated mucin-secreting carcinomas was 6-fold in the skin-wounding DMH-treated group than in the unwounded group and the majority of tumors were located near to lymphoid aggregates. The present results suggest that wound healing enhances tumor development at a distant site, such as the colon, and this effect seems to be related to tumor histology.

SUBJECT HEADINGS: Wound healing. Colonic neoplasms. 1,2 dimethylhydrazine. Rats. Carcinogens.

INTRODUCTION

Many studies have related surgical procedures with enhancement of tumor growth, recurrence and development of metastasis ^{1, 2, 3, 4}. It has been suggested that noncancer operations away from the primary tumor may enhance the growth of an occult tumor in animals or patients ^{5, 6, 7}.

Some experimental models have shown that removal of neoplastic or healthy tissue induces cell proliferation in the remaining tumors ^{3, 13}. Since detectable cytokinetic changes are of short duration, an important question to be clarified is the relevance of the process of repair for tumor progression.

Few data are avaiable on the remote influence of repair and/or regeneration on growth of tumors at a distant site from wounding. The studies were restricted to a limited number of tumor models that have used transplantable tumors innoculated subcutaneously or intraperitoneally ^{10, 7} .

On this basis, we chose to study the remote effect of healing of a full thickness skin wound on tumor development at a distant site, in a model of colon carcinogenesis induced by DMH in the rat.

METHODS

Male Wistar rats, 6-weeks wold, weighing about 160g, were housed in individual wire-mesh cages, in a temperature-controlled room (21-25^oC ) with a 12hr light/dark cycle. They were given food ( Nuvilab-CR, Nuvital,Brazil) and tap water ad libitum. After 10 days of acclimation they were randomly assigned into 4 groups. Two groups of 10 rats received subcutaneous injections of DMH ( 20mg/Kg body weight ) once a week, for eight weeks (DMH- initiated groups). Two control groups of 10 rats received eight weekly injections of PBS plus the vehicle EDTA. DMH was purchased from Sigma Chemical Company (St Louis, MO ). It was dissolved immediately before use in 1mM EDTA and brought to pH 6.5. (Fig. 1)

Figure 1.
Experimental Design

Surgical Procedures

At the 9th week one DMH-initiated group (n=10 ) and one control group (n = 5 ) received a full thickness wound ~ 4cm long in their dorsal skin. For this procedure, animals were lightly anesthetized with ethyl ether and the dorsal skin was cut with surgical scissors to produce a 4 cm full thickness sagital wound. The wound was left to heal by second intention and healing was complete around 12 to 14 days. All groups were sacrificed by ethyl ether inhalation, after the wounds had sufficiently healed at the 12th week. This protocol was based in guidelines of the Commitee on Care and Use of Laboratory Animals of the National Research Council.

Histological analysis

At necropsy the entire colon was removed, opened longitudinally, rinsed in saline and fixed flat on a filter paper in10% buffered formalin. Cecum and rectum were excised and the remaining colon was divided into two segments, the proximal colon near to cecum and distal colon near to rectum. The presence of tumors was recorded. Each segment was rolled as "Swiss roll"and processed for paraffin embedding, sectioning at 4-5 um and staining with hematoxylin and eosin. The neoplasms were classified according to the histopathological classification proposed by Sunter et al ⁹.

Statistical Analysis

Tumor incidence was evaluated for significance by the Fisher exact method. The differences in tumor multiplicity and histology were determined by Student^, s t- test

RESULTS

The animals submitted to the full thickness skin wound presented weigh loss during the first postoperative week, gaining weigh thereafter and showing no significant differences when compared to the other groups throughout the experiment. Healing was complete within 12 to 14 days, with no signs of local infection

The effects of skin wounding on the development of colon tumors induced by DMH are summarized in Table 1.

The number of tumor-bearing rats did not differ among the groups but the total number of tumors and the number of tumors per rat was significantly higher in the skin-wounding DMH-initiated group than in the unwounded group.

Morphological analysis

In the histopathological analysis of the colon the number of poorly differentiated mucin-secreting carcinomas was 6-fold in the skin-wounding DMH-initiated group than in the unwounded group (Table1). These tumors showed endophytic growing pattern and extended into the submucosa in both groups. All poorly differentiated carcinomas were located in the proximal colon and the majority of the lesions were associated with lymphoid aggregates in both groups (Figure 2). The number of well-differentiated carcinomas was also higher in the skin-wounding group (Table 1). The majority of tumors were early intramucosal lesions, characterized as having severe cytological displasia and distorted tubular architecture ( Figure 3). The tumors were located mainly in the distal colon in both groups and no relationship with lymphoid aggregates was observed for this tumor type.

Figure 2
- Poorly-differentiated adenocarcinoma showing signet-ring cells in a rat exposed to eight injections of 1,2 dimethylhydrazine, that received a full thickness skin wound. Hematoxylin and eosin X 400.

Figure 3
- Well differentiated tubular adenocarcinoma in a rat exposed to eight injections of 1,2 dimethylhydrazine, that received a full thickness skin wound. Hematoxylin and eosin X 400.

DISCUSSION

Skin wounding is an established promoting stimulus in studies of epidermal carcinogenesis ^{1, 9}. In the present experiment we produced an ample full thickness skin wound that required restoration of a large amount of tissue for resolution. Our purpose was to generate a strong local proliferative stimulus, with systemic release of factors that might influence the growth of DMH- initiated colonic cells. Data presented in Table 1 demonstrate that although no significant difference on tumor incidence was found between the groups, the number of tumors per rat was significantly higher in the skin-wounding DMH-initiated group than in the unwounded group. These results indicate that skin wounding can exert an enhancing influence on the carcinogenic process at a distant site, such as the colon. The present results suggest that serum factors delivered during the wound-healing process can act systemically at distant sites, inducing chemically initiated cells to grow into a tumor. Our results are in agreement with those reported previously by Gutman et al in a transplantable tumor model ¹⁰. Using human colon cancer cells innoculated subcutaneously in athymic nude mice that underwent partial hepatectomy, the authors showed accelerated tumor growth that directly coincided with liver regeneration.

In the present study, the histopathological analysis of the colon showed that the number of poorly differentiated mucin-secreting carcinomas was 6-fold in the skin-wounding DMH- initiated group than in the unwounded group. This finding suggests that enhancement of tumor growth may be related to tumor histology. It is possible that cells of poorly differentiated carcinomas express higher levels of surface receptors for growth-stimulating factors that are produced during wound healing. Interestingly, the majority of poorly differentiated carcinomas were located near to lymphoid aggregates, as previously registered ¹³. Hardman and Cameron have found higher proliferative activity in the colonic crypts located over lymphoid aggregates in DMH- treated rats and they localized immunohistochemically TGF alfa in the epithelial cells of the proliferative zone ¹³. It is likely that factors produced by cells in the lymphoid aggregates may have a local promoting effect on the development of poorly differentiated carcinomas, that was amplified by circulating growth factors involved in wound healing.

CONCLUSION

This study supports the hypothesis that wound healing can stimulate tumor development at a distant site and this may be related to the sistemic effects of growth- stimulating factors that are produced during the process of repair.

Sequeira JL, Kobayasi S, Rodrigues MAM. Cicatrização e carcinogênese do colon: influência da cicatrização cutânea no modelo de desenvolvimento de tumores do colon induzido pela 1,2 dimetil-hidrazina no rato. Acta Cir Bras [serial online] 2000 Jul-Sept;15(3). Available from: URL: http://www.scielo.br/acb.

RESUMO: Este estudo foi desenvolvido com a finalidade de investigar a possível influência do processo de cicatrização sobre o desenvolvimento neoplásico à distância, em um modelo experimental de carcinogênese do colon induzido pela 1,2 dimetil-hidrazina ( DMH). Ratos Wistar machos receberam injeções semanais de DMH ( 20mg/Kg, via subcutânea) ou solução salina, durante oito semanas. Na nona semana, um grupo tratado com DMH e um controle, foram submetidos à intervenção cirúrgica para retirada de um retalho cutâneo de 4cm no flanco direito, que cicatrizou por segunda intenção. Na 12^a semana, logo após o fechamento da ferida cutânea, os animais foram submetidos à eutanásia. O colon foi dividido em segmentos proximal e distal e examinado a nível macroscópico e histológico. Foram analisadas a incidência, distribuição e morfologia das lesões. O número total de tumores na mucosa do colon e o número de tumores por animal foi significantimente maior no grupo submetido à ferida cutânea do que no grupo tratado somente com DMH. O número de carcinomas pouco diferenciados foi significantimente maior no grupo com ferida cutânea do que em seu respectivo controle. Estes resultados sugerem que o processo de reparação de uma ferida cutânea favorece o desenvolvimento neoplásico em um órgão à distância, tal como o colon e que este efeito parece estar relacionado ao tipo histológico da neoplasia.

DESCRITORES: Cicatrização de feridas. Cólon/Neoplasias. 1,2 dimetil-hidrazina. Ratos. Carcinógenos.

Adress for correspondence:

M A M Rodrigues

Departamento de Patologia, Faculdade de Medicina de Botucatu

Universidade Estadual Paulista

Caixa Postal 564

Botucatu - SP.

18618-000

Fax: [phone]

e-mail: mariar@ fmb.unesp.br

Data do recebimento: 22/05/2000

Data da revisão: 30/06/2000

Data da aprovação: 16/07/2000

2. Prof. Dr Depto Patologia, Fac Med Vet Botucatu, UNESP.

3. Prof Titular Disciplina de Gastroenterologia Cirúrgica, Depto Cirurgia e Ortopedia, Fac Medicina Botucatu, UNESP.

4. Prof Livre Docente, Depto Patologia, Fac Medicina Botucatu, UNESP.

• 1. Bouvy ND, Marquet RL, Jeekel H, Bonjer HJ. Impact of gas(less) laparoscopy and laparotomy on peritoneal tumor growth and abdominal wall metastases. Ann Surg 1996;224:694-700.
• 2. Hofer S, Shrayer D, Reichner JS, Hoekstra HJ, Wanebo HJ. Wound-induced tumor progression: a probable role in recurrence after tumor resection. Arch Surg 1998;133:383-89.
• 3. Jacobi C, Ordemann J, Böhm B, Zieren HU, Liebenthal C, Volk HD, Müller JM. The influence of laparotomy and laparoscopy on tumor growth in a rat model. Surg Endosc 1997;11:618-21.
• 4. Van Dierendonck JH, Keijzer R, Cornelisse CJ, Van de Velde JH. Surgically induced cytokinetic responses in experimental rat mammary tumor models. Cancer 1991;68:759-67.
• 5. Nordlinger B, Quilichini A, Parc R, Hannoun L, Delva E, Huguet C. Hepatic resection for colorectal liver metastasis: influence on survival of preoperative factors and surgery for recurrence in 80 patients. Ann Surg 1987;205:256-63.
• 6. Picardo A, Karpoff HM, Ng B, Lee J, Brennan MF, Fong Y. Partial hepatectomy accelerates local tumor growth: potential roles of local cytokine activation. Surgery 1998;124:157-64.
• 7. Weese JL, Ottery FD, Emoto SE. Do operations facilitate tumor growth? An experimental model in rats. Surgery 1986;100:273-7.
• 8. Fisher B, Gunduz N, Coyle J, Rudock C, Saffer E. Presence of growth- stimulating factor in serum following primary tumor removal in mice. Cancer Res 1998;49:1996-2001.
• 9. Sunter JP, Appleton DR, Wright NA, Watson AJ. Pathological features of the colonic tumours induced in rats by the administration of 1,2-dimethylhydrazine. Virchows Arch Cell Pathol 1978;29:211-23.
• 10. Gutman M, Singh RK, Price JE, Fan D, Fidler IJ. Accelerated growth of human colon cancer cells in nude mice undergoing liver regeneration. Invasion Metastasis 1994;14:362-71.
• 11. Argyris TS. Regeneration and the mechanism of epidermal tumor promotion. Crit Rev Toxicol 1985;14:211-58.
• 12. Van den Tol PM, Van Rossen EE, Van Eijck CH, Bonthuis F, Marquet RL, Jeekel H. Reduction of peritoneal trauma by using nonsurgical gauze leads to less implantation metastasis of spilled tumor cells. Ann Surg 1998;227:242-8.
• 13. Hardman WE, Cameron IL. Colonic crypts located over lymphoid nodules of 1,2-dimethylhydrazine-treated rats are hyperplastic and at high risk of forming adenocarcinomas. Carcinogenesis 1994;15:2353-61.

1

. Parte da Tese de Doutorado apresentada pelo primeiro autor ao Curso de Pós-Graduação em Patologia, Faculdade de Medicina de Botucatu, UNESP.

Publication Dates

• Publication in this collection
  04 Sept 2000
• Date of issue
  Sept 2000

History

• Accepted
  16 July 2000
• Reviewed
  30 June 2000
• Received
  22 May 2000