Effects of dietary lipids on cell proliferation of murine oral mucosa

1476-511X-1-3 1476-511X Research Effects of dietary lipids on cell proliferation of murine oral mucosa Actis AB adractis@odo.unc.edu.ar Joekes S joekess@eco.uncor.edu Cremonezzi D dccj35@yahoo.com.ar Morales G gustavomor@yahoo.com Eynard AR aeynard@cmefcm.uncor.edu

Instituto de Biología Celular, Facultad de Ciencias Médicas. Universidad Nacional de Córdoba. 5000 Córdoba, Argentina

Instituto de Estadística y Demografía, Facultad de Ciencias Económicas. Universidad Nacional de Córdoba. 5000 Córdoba, Argentina

Lipids in Health and Disease 1476-511X 2002 1 1 3 http://www.Lipidworld.com/content/1/1/3 10.1186/1476-511X-1-3 12617749 15 10 2002 20 11 2002 20 11 2002 2002 Actis et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. cell proliferation dietary lipids PUFAs fatty acids oral mucosa

Abstract

Background

The lack of certain essential polyunsaturated fatty acids (PUFAs) induces perturbation in cell proliferation, apoptosis and dedifferentiation that could be linked to an increased protumorigenic trend. Contrarily, n-3 essential fatty acids (EFAs) arrest cell proliferation in several tumor models. According to the concept of field cancerization, multiple patches of abnormal epithelial proliferation may coexist in the vicinity of oropharyngeal neoplasms.

The purpose of the present study is to determine whether certain dietary PUFAs differentially modulate the patterns of cell proliferation and apoptosis at non-tumoral sites of the oral mucosa in mice bearing DMBA induced salivary tumors.

After weaning, BALB/c mice were assigned to four diets: Control (C), Corn Oil (CO), Fish (FO) and Olein (O). Two weeks later, DMBA was injected into the submandibular area. The animals were sacrificed between 94 and 184 days at 4–6 PM. Fixed samples of lip, tongue and palate were stained using H-E and a silver technique. A quantification of AgNORs in the basal (BS) and suprabasal stratum (SBS) of the covering squamous epithelia as well as of mitosis and apoptosis was performed.

Results

Analysis of Variance showed greater proliferation in tongue than in palate or lip. According to the diet, a significant difference was found in the Fish Oil, in which palate exhibited fewer AgNOR particles than that of the control group, both for BS and SBS (p < 0.05 and 0.152, respectively), indicating a reduced cell proliferation.

Conclusions

These results corroborate and reaffirm that the patterns of cell proliferation, apoptosis and differentiation of the oral stratified squamous epithelium may be differentially modulated by dietary lipids, and arrested by n-3 fatty acids, as shown in several other cell populations.

Background

Oral mucosa has a squamous stratified epithelium with four cell layers, namely: basal, malpighian, granular and corneum. Proliferation, which takes place in the basal stratum, along with differentiation and apoptosis must be in balance due to the permanent cell renovation.

Essential fatty acids (EFAs) such as linoleic acid (n-6 LA) and α-linolenic acid (n-3 ALA) are food lipids obtained only from the diet. Cell proliferation, differentiation and apoptosis was shown to be modulated by n-3 and n-6 EFAs in different tissues 310. Contrarily, the essential fatty acid deficiency (EFAD) induces phenomena of cell proliferation and dedifferentiation along with abnormal apoptosis that could be linked to an increased protumorigenic trend. These facts were observed in skin, oral epithelium, esophagus, salivary glands, urothelium and intestine of rats. 4512141518.

The modulating effects of EFAs have been found not only in normal but also in neoplastic tissues, such as breast, colon and urothelium 311. Recent experimental studies have shown that n-3 fatty acids exert a protective effect on murine salivary gland tumors, whereas n-9 olein enriched diet, which induces an EFAD condition, has a promoting activity 2.

Molecular and clinical evidence support the phenomenon of field cancerization for head and neck tumors 813. Some varieties of salivary gland tumor proliferation may coexist with oropharyngeal neoplasms 17. They may also adopt a biphasic differentiation pattern 67 and develop adenocarcinomas or epidermoid carcinomas, the latter arising from their epithelial components 9.

Materials and methods

Animals and dietary conditions

Forty-eight BALB/c mice of both sexes were randomly assigned, after weaning, to four dietary groups: Control (C), Corn Oil (CO), Fish (FO) y Olein (O). The first was fed on a conventional diet containing 5% mixed fat, 20% protein, 67% carbohydrates and 2 % fiber. The rest received a basic semisynthetic formula containing: casein 17%; sucrose 34.9%; corn starch 38%; fiber 2%; salt mixture 3.5%; vitamin mixture 0.5% 2. This preparation was supplemented with 5% corn oil (CO), fish oil (FO) or double refined olein (O) (80% of 18:1 n-9; Retienne, Argentina), enriched in n-6, n-3 and n-9, respectively. These diets, which were stored in sealed dark containers at -5°C, did not significantly differ in caloric value per gram weight. Food and water were provided ad libitum to the animals, which were kept in a light and temperature controlled room. Fatty acid composition of dietary formula is shown in Table 1.

Table 1

Fatty acid composition of dietary fats.

Diet

Saturated

Mono-UFAs

PUFAs

16:0

18:0

20:0

16:1 n-7

18:1 n-9

18:2 n-6

18:3 n-6

18:3 n-3

20:5 n-3

22:5 n-3

22:6 n-3

25.4

8.1

1.7

1.5

41.2

10.1

3.3

13.5

4.6

1.1

0.6

33.1

47.1

12.7

2.5

3.4

9.4

35.0

2.6

1.5

5.3

4.8

10.4

3.6

0.3

7.6

79.5

2.9

0.6

1.2

Mono-UFAs: monosaturated fatty acids; PUFAs: polyunsaturated fatty acids; tr: traces. * Control (C), Corn oil (CO), Fish oil (FO), Olein (O). Values represent percentage mean of each fatty acid for no less than three samples run in duplicate.

Two weeks later, 0.5 mg 9,10-dimethyl-1,2-benzanthracene (Sigma) diluted in 1 μl of corn oil was subcutaneously injected into the submandibular area. The animals, which developed histologically confirmed salivary tumors in 100% of cases, were sacrificed between 94 and 184 days at 4–6 p.m. The study followed the protocol established for salivary gland tumors 2 and the whole head was randomly taken from 16 mice to carry out the present analysis.

Histological procedures

The heads were fixed in 10% neutral formalin and samples of lip, tongue and palate were obtained. They were embedded in paraffin, sliced perpendicularly to the mucosa so as to observe all the constitutive strata and 3–4 μm non-serial sections were stained using Hematoxilin-Eosin (H-E) and the Ploton's silver technique for AgNORs 16.

Microscopic procedures

By using an Olympus BH2-DO light microscope at 400×, a quantification of AgNORs (Nucleolar Organizer Regions) in the basal (BS) and suprabasal stratum (SBS) as well as of mitosis and apoptosis was performed on silver-stained and H-E sections, respectively.

The microscopic examination was carried out by two different observers who were unaware of the dietary conditions of animals. A total of 100 cells were counted (50 in BS and 50 in SBS) for AgNORs, whereas mitosis and apoptosis were recorded for 20 fields (Fig. 1). Small cells with fragmented pycnotic nuclei and eosinophilic cytoplasm surrounded by vesicles were considered apoptotic 3 (Fig. 1-D).

Figure 1

A: Dorsum of tongue, Malpighian layer

A: Dorsum of tongue, Malpighian layer. Silver Nitrate staining for AgNOR bodies within the nucleus (arrow). 400×. Olein supplemented diet. B: Dorsum of tongue, Malpighian layer. Fish oil enriched diet. Fewer AgNOR particles are observed (arrow). C: Malpighian layer of lip. 400×. H-E. Several mitosis are shown. Olein supplemented diet. D: Squamous epithelium of the dorsal aspect of tongue with one apoptotic figure (arrow). 400×. H-E. Corn oil supplemented diet.

Results

Means were calculated from the data of the observers for palate, tongue and lip. The Analysis of Variance was applied to compare localization and diets. Through AgNOR and mitosis quantification, the tongue epithelium presented greater proliferation than palate and lip, both in Fish and Corn Oil groups (Table 2). According to the dietary formula, a significant difference was found in Fish Oil group (rich in n-3 family of PUFAs), in which palate exhibited fewer AgNOR particles than that of the control group, both for BS and SBS (p < 0.05 and 0.152, respectively), indicating a reduced cell proliferation) (Fig. 2).

Figure 2

Number of AgNOR in palate basal and suprabasal strata according to diet

Number of AgNOR in palate basal and suprabasal strata according to diet.

Table 2

Cell proliferation and apoptosis according to localization and diet.

Fish Oil

Corn Oil

Tongue > Palate

p < 0.145

-----

SBS

Tongue > Palate

p < 0.126

Tongue > Lip

p < 0.134

----

Mitosis

Palate > Lip

p < 0.056

Tongue > Palate

p < 0.125

Tongue > Lip

p < 0.083

Apoptosis

----

Tongue > Lip

p < 0.024

Discussion and conclusions

Among other mechanisms, tumor progression involves non-arrested mitotic activity followed by inflammatory infiltration and necrosis. These cellular events make the morphological evaluation of mitotic and apoptotic figures difficult. So, considering that the subcutaneous injection of DMBA may also affect the surrounding areas, we analyzed certain non-tumoral, apparently normal tissues. This approach fits in well with the concept of field cancerization 813.

Present results corroborate and reaffirm that the particular patterns of cell proliferation and differentiation of the oral stratified squamous epithelium may be modulated by dietary lipids 5. In particular, n-3 fatty acids comparatively diminished the hyperproliferative effect linked to DMBA administration. The beneficial effects of n-3 rich food should be emphasized in the prevention of oral neoplastic lesions, as previously proposed 12.

Authors' contributions

ABA and ARE conceived of the study and participated in its design and coordination as well as in the experimental pathology. DC carried out the microscopic examination. GM applied the histologic techniques. SJ participated in the statistical analysis.

Influence of environmental and nutritional factors on salivary gland tumorigenesis, with special reference to dietary lipids. Actis AB Eynard AR Eur J Clin Nutr 2000 54 805 810 10.1038/sj.ejcn.1601077 11114673 N-3, n-6 and n-9 dietary fatty acids modulate the growth parameters of murine salivary gland tumors induced by dimethylbenzanthracene. Actis AB López CB Joekes S Eynard AR Prostagl Leuk Essent Fatty Acids 1999 61 259 265 10.1054/plef.1999.0099 Dietary polyunsaturated fatty acids (PUFA) differentially modulate melamine-induced pre and neoplastic urothelial proliferation and apoptosis in mice. Cremonezzi DC Silva RA Díaz MP Eynard AR Prostagl Leuk Essential Fatty Acids 2001 64 151 159 10.1054/plef.2001.0255 Cis-unsaturated fatty acids as potential anti-mutagenic, tumoricidal and anti-metastatic agents. Das UN Asia Pacific J Pharmacol 1992 7 305 327 Increased proliferation of the epithelium of the proximal alimentary tract of EFA-deficient rats: a light and electron microscopic study. Eynard AR Monis B Kalinek F Leguizamón RO Exp Mol Pathol 1982 36 135 143 7060719 Ultrastructure of sialadenoma papilliferum. Fantasía JE Nocco CE Lally ET Arch Pathol Lab Med 1986 110 523 527 3754726 Sialadenoma papilliferum. Report of two cases. Freedman PD Lumerman H Oral Surg Oral Med Oral Pathol 1978 45 88 94 271294 Photodynamic therapy of malignant and premalignant lesions in patients with "field cancerization" of the oral cavity. Grant WE Hopper C Speight PM Macrobert AJ Bown SG J laryngol Otol 1993 107 1140 1145 8289004 Squamous cell carcinoma arising in Warthin's tumor of the parotid gland. A light, electron microscopic and immunohistochemical study. Jamjanov I Sneff EM Delerme AN Oral Surg Oral Med Oral Pathol 1983 55 286 290 6188086 Essential fatty acids: molecular and cellular basis of their anticancer action and clinical implications. Jiang WG Bryce RP Horrobin DF Critical Rev Oncol Hematol 1998 27 170 209 Polyunsaturated fatty acids and their role in cancer invasion and metastasis. Cancer Metastasis, Molecular and Cellular Mechanisms and Clinical Intervention. Jiang WG Redfern A Eynard AR Mansel RE Kluwer Academic Publishers, Netherland Jiang WG, Mansel RE 2000 225 250 10932001 Essential fatty acid deficient hairless mouse: a model of chronic epidermal hyperproliferation. Lowe NJ Stoughton RB Br J Dermatol 1977 96 155 162 843450 Molecular support for field cancerization in the head and neck. Lydiatt WM Anderson PE Bazzana T Casale M Hughes CJ Huvos AG Lydiatt DI Schantz SP Cancer 1998 82 1376 1380 10.1002/(SICI)1097-0142(19980401)82:7<1376::AID-CNCR22>3.0.CO;2-2 9529031 Abnormal cell proliferation and differentiation and urothelial tumorigenesis in essential fatty acid deficient (EFAD) rats. Monis B Eynard AR Prog Lipid Res 1981 20 691 703 10.1016/0163-7827(81)90127-2 7342122 Mitotic activity in the skin of mice deficient in essential fatty acids. Nasr AN Shostak S Nature 1965 207 1395 5886044 Improvement in the staining and visualization of argyrophilic proteins of the nucleolar organiser region at the optical levels. Ploton D Menager M Jeannesson P Himber G Pigeon E Adnet J Histochem J 1986 18 5 14 2423479 Multicentric Warthin's tumor of the paraparotid region mimicking limph node metastases of homolateral oral and oropharyngeal squamous cell carcinoma: reports of two cases. Sato T Ishibashi K J Oral Maxillofac Surg 1998 56 75 80 9437986 The effect of essential fatty acid deficiency on the ultrastructure and functional capacity of the jejunal epithelium. Snipes RL Lab Invest 1968 18 179 189 5644532