Ultrastructural changes of the palatal mucosa following application of 4-nitroquinoline-l-oxide (4NQO) in rats subjected to major salivary gland excision.

OBJECTIVE: It has been suggested that saliva exerts a protective role against the carcinogenic effect of various substances in the oral cavity. The objective of this study was to examine the ultrastructural changes of the palatal mucosa caused by the application of 4-nitroquinoline-l-oxide (4NQO) in the presence or absence of saliva. STUDY DESIGN: Wistar-Furth rats subjected and not subjected to total bilateral excision of the major salivary glands were either painted with an aqueous solution of 4NQO or with propylene glycol only (controls). Two animals of each group were humanely killed periodically. The areas of the palatal lesions were immediately sliced and processed for TEM examination. RESULTS: Ultrastructurally, the progressive changes to squamous cell carcinoma were observed in the animals painted with 4NQO. In the desalivated animals group, the ultrastructural alterations appeared earlier than in the group with salivary glands. CONCLUSIONS: Saliva appeared to delay but not hinder tumor induction by 4NQO.

In vivo photo-detection of chemically induced premalignant lesions and squamous cell carcinoma of the rat palatal mucosa.

Photo-detection using in vivo fluorescence was studied for different stages of chemically induced premalignant lesions and squamous cell carcinoma (SCC) of the Wistar rat palatal mucosa. It was found that the epithelial dysplasia (numerically expressed in the epithelial atypia index (EAI) of the rat palate, induced by repeated application of the carcinogen 4-nitroquinoline 1-oxide (4NQO), showed an increase approximately proportional to the duration of the application period. Photo-detection of the lesions using Photofrin-induced fluorescence was studied with dual-wavelength excitation and the subtraction of images, in an attempt to reduce the autofluorescence. The Photofrin dose was 2.5 mg kg-1. This was based on a dose-response study for normal tissue damage by photodynamic therapy (PDT) in this animal model, because the underlying rationale was to study photo-detection as a method of locating additional (early) malignancies in patients treated by PDT. Fluorescence intensities 24 and 48 h after injection of Photofrin were shown to increase with the duration of 4NQO application and with increasing EAI. For an EAI greater than 15, there was a statistically significant difference (p < 0.01) between the fluorescence signals obtained with and without the injection of Photofrin. Fluorescence signals of these lesions without the use of Photofrin (autofluorescence) also showed an increase with increasing stages of epithelial dysplasia of the rat palate. However, the fluorescence signals obtained with Photofrin were always higher than those of the autofluorescence. From this study, we conclude that photo-detection with Photofrin has potential in distinguishing chemically induced premalignant lesions and squamous cell carcinomas from the normal rat palatal mucosa. Photofrin (2.5 mg per kg of body weight) certainly adds to the sensitivity of photo-detection, but autofluorescence alone also has promising features for detecting premalignant and malignant lesions of the oral mucosa.

Photofrin-mediated photodynamic therapy of chemically-induced premalignant lesions and squamous cell carcinoma of the palatal mucosa in rats.

Photodynamic therapy (PDT), an experimental cancer therapy, was studied in an animal model of chemically-induced epithelial dysplasia and squamous cell carcinoma. PDT was performed 24 hours after i.v. injection of 2.5 mg/kg bw Photofrin, and using 100 J/cm2 incident light at two activation wavelengths (514.5 nm or 625 nm). Two days after PDT, the majority of rats macroscopically showed a marked erythema of the entire palatal region. Microscopically all the rats showed oedema, haemorrhage, and necrosis of the epithelium of the intermolar area. The long-term results were not so favourable. No evidence of disease was found in 6 out of 20 rats in the 514.5 nm group and in 2 out of 20 rats in the 625 nm treated group. Epithelial dysplasia was found in 14 out of 20 rats in the 514.5 nm group, and in 18 out of 20 rats of the 625 nm treated group. Squamous cell carcinomas were found in 4 out of 20 rats treated with 514.5 nm and in 7 out of 20 rats in the 625 nm treated groups. Comparing both treatment wavelengths, better results were obtained in the 514.5 nm groups as this wavelength gave less normal tissue damage. Based on the results of this study the application of PDT for the treatment of field cancerization and squamous cell carcinoma of the oral cavity, is discussed.

Epithelial dysplasia and squamous cell carcinoma of the Wistar rat palatal mucosa: 4NQO model.

BACKGROUND: The carcinogen 4-nitroquinoline 1-oxide (4NQO) has been used in several studies concerning experimental oral carcinogenesis to induce squamous cell carcinoma in the palatal mucosa of rats, whereas limited attention has been paid to preceding premalignant mucosal changes. The aim of this study was to describe the macroscopic and microscopic changes of the rat palatal mucosa treated with 4NQO as a function of the application time of this carcinogen. METHODS: The carcinogen 4NQO was applied to the palatal mucosa of male Wistar rats three times a week for 2-26 weeks. The Epithelial Atypia Index (EAI) was used to quantify the degree of dysplasia induced by the application of 4NQO for various time intervals. RESULTS: This study shows that the EAI increases as a function of the application time. As soon as 2 weeks following application of the carcinogen, dysplastic alterations in the mucosa were observed. The extent and grade of the dysplastic changes increased with time. At week 26, squamous cell carcinoma developed. In rats in which the application of 4NQO was discontinued earlier, the application period appeared to be in inverse proportion to the latency period i.e., the period between the last application of the carcinogen and the first clinical sign of squamous cell carcinoma. This emphasizes the premalignant character of the induced epithelial changes, because in all the rats squamous cell carcinoma eventually developed. CONCLUSIONS: From this study, we concluded that the 4NQO rat palate model is a proper model in which both premalignant epithelial lesions and squamous cell carcinoma of the oral mucosa can be studied.

In vivo fluorescence kinetics and localisation of aluminum phthalocyanine disulphonate in an autologous tumour model.

Sulphonated phthalocyanines are studied as photosensitizers for photodynamic therapy of cancer. Their strong fluorescence and tumour-localising properties make them also potentially useful for detection of cancer by fluorescence. For this purpose, we have studied the fluorescence kinetics and localisation of aluminum phthalocyanine disulphonate (AlPcS2) in 4-nitroquinoline 1-oxide (4NQO)-induced dysplasia and invasive cancer of the oral mucosa of the hard palate in Wistar albino rats. Twenty-two rats were divided into six groups. Five groups were subjected to a 4NQO application period of 8, 12, 16, 20 or 26 weeks and one was a control group. The dysplasia varied from slight to severe and was correlated with the duration of the application period. All animals received a dose of 1 micromol/kg AlPcS2 i.v. Fluorescence images were recorded via a specially designed 'palatoscope' with excitation at 460 +/- 20 nm for autofluorescence, 610 +/- 15 nm for AlPcS2 fluorescence and detection of emission at 675 +/- 15 nm. After subtraction of the two images the specific AlPcS2 fluorescence remained. AlPcS2-mediated fluorescence increased significantly when the severity of dysplasia increased (P<0.04). Also the phenomenon of strong fluorescent spots on the fluorescence images was observed. This always occurred within the first 10 h after injection of AlPcS2. Histological analysis showed a local alteration to a mucosa in 67% of these spots, which was either invasive cancer (29%) or inflammation (38%). These results suggest two different mechanisms of AlPcS2 uptake in tissue, one associated with the presence of generalised dysplasia and another associated with local changes of the epithelial/connective tissue, which is not necessarily specific for tumours.

4NQO carcinogenesis: a mouse model of oral cavity squamous cell carcinoma.

BACKGROUND: A murine model of oral cavity carcinogenesis is needed to study the molecular aspects of malignant transformation. 4-Nitroquinoline-1-oxide (4NQO), a water-soluble carcinogen, produces squamous cell carcinoma in rodents. Protocols were designed to investigate the temporal aspects of neoplastic transformation. METHODS: 4NQO was applied topically to mouse palates for up to 16 weeks. Mice were observed and killed from 24 to 49 weeks. RESULTS: A spectrum of lesions ranging from atypia to moderately differentiated invasive squamous cell carcinoma (SCC) was produced. The severity of the lesions corresponded to the duration of treatment and the length of observation. There was no gross or microscopic evidence of an inflammatory reaction to 4NQO. The lesions were focal and normal mucosa predominated in the treated mice. CONCLUSION: 4NQO reliably produced preneoplastic and malignant oral cavity lesions, which morphologically and histologically mimic human head and neck cancer. Lesions develop long after 4NQO exposure and without an inflammatory response. Thus, the model should be useful for molecular analysis of neoplastic transformation.

Candida albicans as a promoter of oral mucosal neoplasia.

A model of oral mucosal carcinogenesis using the water-soluble carcinogen 4-nitroquinoline-1-oxide (4NQO) was combined with a model of oral mucosal candidosis to examine the ability of Candida albicans to promote the development of neoplasia in suitably initiated epithelium. Sprague-Dawley rats were initiated by the application of 4NQO to their palatal and tongue mucosa 3 times weekly for 4 weeks. The animals then received either application of a phorbol ester to act as a promoter, induction of experimental oral mucosal infection with C. albicans, or no further procedures. Animals were killed at 34 or 52 weeks and the tongues and palates sectioned for light-microscopic examination. Control groups with no treatment, mucosal infection only, phorbol ester application only, 4NQO with the tetracycline or vehicle application only were also used. The development of carcinoma in the experimental groups was similar to that in the positive control groups, indicating that the particular strain of Candida used had a similar ability to promote neoplastic changes as the known promoter phorbol-12,13-didecanoate and caused neoplastic changes to occur by week 34 with no additional lesions occurring by week 52. This indicated that the speculation that strains of C. albicans may participate in causing neoplastic transformation in humans was well founded.

The development of B-cell lymphoma in a patient with psoriasis treated with cyclosporine.

A 67-year-old Nicaraguan man with a history of chronic, severe, recalcitrant psoriasis participated in a multicenter study investigating oral cyclosporine in the treatment of psoriasis. He received cyclosporine for approximately 8 months at doses of 5 mg/kg/day or less. Treatment with cyclosporine was eventually discontinued because of progressive nephrotoxicity. Approximately 7 months after the discontinuation of cyclosporine, the patient developed a mass in the left maxillary sinus extending to the orbit, the palate, and the infratemporal fossa. Pathologic and histochemical analysis of the mass revealed a B-cell lymphoma. The development of a benign lymphocytic infiltrate has been reported in a patient who received cyclosporine therapy for psoriasis; however, to the best of our knowledge, this is the first case in the United States of lymphoma developing in a patient who was treated with cyclosporine for a condition other than organ transplantation.

A relationship found between intra-oral sites of 4NQO reductase activity and chemical carcinogenesis.

Topical application on rat oral mucosa of the chemical 4-nitroquinoline 1-oxide (4NQO) has been shown to produce squamous cell carcinomas on the posterior tongue and/or the posterior hard palate. 4NQO is broken down in vivo by a diaphorase, 4NQO reductase (E.C.1.6.99.2), to produce an active molecule believed to be responsible for carcinogenesis. It has been shown that there are higher concentrations of 4NQO reductase in oesophageal mucosa compared with elsewhere in the gastrointestinal tract. The purpose of these experiments was to compare the distribution of certain diaphorases in the oral mucosa. Samples of rat tongue and cheek epithelia were homogenized, then ultracentrifuged to provide mixed cytosol and microsome fractions from the epithelial cells. A spectrophotometer was used to measure the variation in absorbance at 340 nm of NADH consumed by reduction of 4NQO by enzymes present in the tissue extracts. A histochemical technique was used to compare the activity of NADH diaphorase, NADP diaphorase and glucose-6-phosphate dehydrogenase at different sites of the oral mucosa. Statistical analysis showed that there were significant (P less than 0.01) differences between the activities of all three enzymes at different sites of the oral mucosa. In each case, a higher activity was found at the sites of high incidence of squamous cell carcinoma. A lower activity was found at sites where carcinomas did not occur.

Effect of oral contraceptive on early DMBA carcinogenesis of the rat palatal mucosa.

Experiments were undertaken to find out whether administration of oral contraceptive (OC) per nasogastric tube could accelerate or inhibit early carcinogenesis of the rat palatal mucosa induced by painting 0.5% solution of 7,12-Dimethyl benz (a) anthracene (DMBA) in liquid paraffin. Forty female Sprague-Dawley rats were divided into 8 groups consisting of 5 animals per group. Group I--DMBA; Group II--DMBA + Oral Contraceptive I (13.0gm% Norethindrone + 8.7mg% ethyl estradiol); Group III--DMBA + Oral Contraceptive II (6.5mg% Norethindrone + 4.3% ethyl estradiol). Group IV--Oral Contraceptive I, Group V--Oral Contraceptive II, Group VI--liquid paraffin, Group VII--Corn oil and Group VIII--untreated control. Each animal was sacrificed at 18 weeks, the palatal mucosa degloved, fixed in 10% formal saline, sectioned in paraffin at 5u, stained with Hematoxylin and Eosin and evaluated histologically for features of epithelial dysplasia. Morphometric analysis was performed on epithelial and keratin thickness. Results indicate higher mean grade of epithelial dysplasia for group II when compared to group I (P less than 0.01) thereby suggesting a cocarcinogenic action for the Oral Contraceptive I. Lower dose Oral Contraceptive II was, however, unable to produce any significant mean grade of dysplasia in group III, suggesting further that the cocarcinogenic action of the Oral Contraceptive used was dose dependent. Morphometric analysis shows a decrease (P less than 0.01) in Keratin and palatal epithelium in DMBA treated animals and further decrease (P less than .001) in those two structures when Oral Contraceptive was used in combination with DMBA, suggesting that the cocarcinogenic action of Oral Contraceptive could result from the induced atrophy of keratin/epithelium and subsequent improved access of the DMBA to target cells in the basal layer of the epithelium. It is suggested that Oral Contraceptive accelerated the induction of early DMBA carcinogenesis of the rat palatal mucosa in this study.

Kaposi's sarcoma in kidney transplant recipients treated with cyclosporin.

We report the clinical and histological features and the course of Kaposi's sarcoma in three transplant patients treated with cyclosporin. Four months after cyclosporin treatment was stopped the lesions in all three patients had completely healed.

Blood group antigen staining pattern during experimental carcinogenesis in rat palate.

During 4-nitroquinoline-1-oxide-induced carcinogenesis in the rat palate, animals were sacrificed at various intervals and stained for blood group antigens B and H (Type 2 chain) by an immunofluorescent method. In rats without signs of epithelial dysplasia, the staining pattern was identical with that in the normal control rats. In rats with definite or questionable (borderline cases) dysplasias, marked changes in blood group antigen staining pattern were seen. Thus, changes in cell-surface carbohydrates during malignant development in the rat palate seem to follow closely the histomorphological changes. As there is good evidence that carcinomas would eventually develop in all rats if they were not sacrificed, it seems that the blood group antigen staining pattern does not predict malignant development in the absence of histological suspicion.

The distribution of blood group antigens in experimentally produced carcinomas of rat palate.

It has been shown previously that rat oral epithelia express antigens cross-reacting with antibodies against human blood group antigen B and its structural precursor, the H antigen (Type 2 chain). In the present study we investigated the expression of these antigens in malignant changes in the rat palate induced by a chemical carcinogen (4NQO). The H antigen, normally expressed on spinous cells in rats, was absent in malignant epithelium, whereas staining for the B antigen, normally expressed on basal cells, was variable. These changes are equivalent to those seen in human squamous cell carcinomas. The blood group antigen staining pattern in experimentally produced verrucous carcinomas showed an almost normal blood group antigen expression. This may have diagnostic significance. Localized areas of hyperplastic palatal epithelium with slight dysplasia revealed loss of H antigen and the presence of B antigen in suprabasal strata equivalent to the pattern seen in human premalignant epithelium. We conclude from these findings, that the rat model is well suited to study changes in cell surface carbohydrates during chemical carcinogenesis.

Verrucous hyperplasia and verrucous carcinoma of the rat oral mucosa. Experimental oral carcinogenesis using 4-nitroquinoline 1-oxide.

Verrucous hyperplasia and carcinoma are recognized entities in the human pathology, but not documented experimentally. During application of the carcinogen 4-nitroquinoline 1-oxide to the oral cavity of rats three times a week for a maximum of 18 weeks an increasing frequency of verrucous hyperplasias and carcinomas were noted. The majority of the carcinomas were localized to the palate and 3/4 of these were verrucous carcinomas, with or without anaplastic transformation to infiltrating squamous cell carcinomas. The oral verrucous lesions have all the histological characteristics of their human counterparts. The present experimental study supports the idea that tobacco consumption may represent an important etiological factor in the development of human, oral verrucous carcinomas.

Experimental induction of oral squamous cell carcinomas in mice with 4-nitroquinolone-1-oxide.

The oral mucosa of mice is resistant to the action of a variety of carcinogens. In this study, the water-soluble carcinogen 4-nitroquinoline-1-oxide (4NQO) was applied repeatedly to the palates of male CBA mice for 2, 4, 6, 8, 12, or 16 weeks, and the animals were observed for the remainder of the 50-week experimental period. Oral epithelial atypia and squamous cell carcinoma were observed with increasing prevalence as the period of carcinogen exposure was increased. Carcinomas developed by 50 weeks in all animals that received 4NQO for 16 weeks.

Electrophoretic study of keratin polypeptides in chemically-induced oral carcinomas in the hamster.

Comparison was made of the electrophoretic keratin polypeptide patterns of normal hard palate epithelia from three hamsters and of eight palatal squamous cell carcinomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) treatment. Keratin polypeptides from normal epithelia had a molecular weight range of about 48,000 to 70,000. In the tumour extracts, the large polypeptides (above 61,000) found in the normal epithelia were absent, but the majority of other small polypeptides below 61,000 were expressed. Three as yet undefined polypeptides, in the range of 40,000 to 70,000, were detected in tumour extracts, but not in extracts of normal palatal mucosa. The keratin polypeptide electrophoretic alterations in carcinomas of hamster palatal mucosa are similar to those reported for extra-oral carcinomas in other animal species.

4 Nitroquinoline 1-oxide-induced carcinogenesis in the rat palate.

Most reported studies of experimental oral cancer have been carried out in the hamster. This animal is not available in certain countries and there is controversy regarding its suitability as a model for experimental oral carcinogenesis. An attempt was made to reproduce an experimental oral cancer model in rats, using a protocol based on that described by Lekholm and Wallenius (1976). The carcinogen 4 nitroquinoline 1-oxide was applied to the palates of rats for periods of up to 24 weeks. Macroscopic and microscopic examination of carcinogen-treated palatal mucosa was carried out during the experimental period. Verrucous carcinoma-like lesions of the mid-palate and squamous carcinomas of the gingival mucosa were produced, beginning at 16 and 20 weeks respectively. A variety of other macroscopic and microscopic mucosal changes were also observed during the experimental period. The findings are discussed in relation to the work of others.

Kaposi's sarcoma in a renal allograft recipient under longterm immunosuppressive therapy.

A 40-year-old Caucasian male with chronic glomerulonephritis received a cadaver renal transplant in July 1973, and received continuous immunosuppressive therapy with Azathioprine and prednisone. His renal function deteriorated during the autumn of 1977, and he developed haemangiomatous tumours in the soft palate and on the sole of the right foot in April 1978. Both were surgically removed and histologically confirmed as Kaposi's sarcomata. The patient died a month later of renal insufficiency. At autopsy, a Koposi's sarcoma was also present in the right lateral malleolar region. There were multiple calcifications in the lungs, vessel walls, striated muscle and subcutaneous fat. To our knowledge, this is the 29th reported case of Kaposi's sarcoma occurring in renal allograft recipients under long-term immunosuppressive therapy. It is concluded that the development of this tumour is probably related to such therapy.