**3.2 Establishment of experimental NPC and further studies**

## **3.2.1 Establishment of the NPC animal model**

After establishing the optimal carcinogens and method of administration, we succeeded in the induction of experimental NPC in rats using DNP [57]. This suggests that chemical carcinogens might be among etiologic factors of NPC. Furthermore, subcutaneous injection of DNP could induce NPC without complications of liver cancer. Therefore, DNP showed organ specificity for nasopharyngeal epithelium. Moreover, DNP-induced NPC exhibited a consistently high incidence rate, thereby paving the way for a subsequent study of DNP in which the carcinogenesis of experimental NPC in rats was further investigated, including atypical cytokinetics in carcinogenesis of the nasopharyngeal epithelium, DNA damage and repair of the nasopharyngeal epithelium by DNP and its relation to tissue specificity, and changes in enzyme activities during carcinogenesis. The results of these studies are discussed below.

#### **3.2.2 Susceptibility of rat nasopharyngeal epithelium to the carcinogens DEN and DMBA**

The induction of NPC in rats by treatment with DEN and DMBA is summarized in Table 1 [72, 73]. These studies showed that the highest incidence of NPC was achieved in the DEN instillation group, and that NPC with less differentiated cells at early stages developed in the rats receiving DEN instillation and DMBA insertion. Occasional lymphatic emboli and multiple metastases to lung were observed. The data suggest some synergic effect between these chemical compounds.

Incidence of NPC in the DEN instillation group was higher than that of the group treated with *s.c.* injection of DEN and empty plastic tube insertion. This suggested that DNE was most effective on nasopharyngeal epithelium. The group with empty tube insertion was examined to exclude any possible carcinogenic action of the polyvinyl chloride tube. The empty tube itself occasionally induced NPC (two cases of NPC in 23 rats). Incidence of NPC in this group was quite lower than other groups [72, 73]. Therefore, it was suggested that NPC development in these groups was mainly caused by the action of carcinogens.


Chemical Carcinogenesis and Nasopharyngeal Carcinoma 67

The data showed that DEN instillation resulted in the highest incidence of NPC. The highest incidence of tumor was in the group treated with DEN and vitamin B12, followed by the group given nitrosomorpholin carcinogen. Two tumors with a nodular and ulcerated appearance were visible to the naked eye, one in DEN group and one in nitrosomorpholin group. Both cases were squamous cell carcinomas, partly involving or arising from the soft palate. These tumors invaded surrounding stroma, nerve bundles, and salivary glands

A few other points were of interest: (a) many squamous carcinomas occurred in neighborhood of auditory pharyngeal tubes or extended along tube walls, indicating that pathological lesions occurred in nasopharyngeal cavity. (b) In nasal cavities of some rats with extensive tumor, the tumor was found only in nasopharyngeal region without invasion of the nasal cavities or the base of the skull. Under naked-eye examination, no tumor was visible in the esophagus or other organs except the liver. (c) Subcutaneous injection of cyclic nitrosamines induced a considerably high incidence of nasopharyngeal tumors, providing some clues to organ specificity of carcinogens. (d) It is worth noting that administration of vitamin B12 markedly increased the incidence of nasopharyngeal cancer (p<0.01), but significantly decreased the incidence of hepatoma. Poirier et al [58] observed that vitamin B12 enhanced hepatic carcinogenesis and shortened the animals' lives. In the present experiments, vitamin B12 increased the incidence of NPC in the DEN group but had the opposite effect in the DNP group. The precise mechanism of this complicated relationship between the carcinogen and nutritional factors needs further study. (e) Vitamin B12, piperazine, and morpholin are usually used in clinics or in pharmaceutical chemistry. Some of them are precursors of the carcinogenic substance and some have an enhancing effect on

Further investigation into experimental induction of NPC showed that N-nitrosomorpholin and DNP could induce NPC in rats, and subcutaneous injection of DNP could induce NPC without complications of liver cancer. These results are summarized in Table 3 [72, 73].

A total of 54 rats developed NPC in this experiment. In addition, 39 cancers of the nasal cavities, 32 esophageal tumors, and a few other tumors were observed. Upon postmortem examination, six rats were found to have gross tumor masses in nasopharynx, and two of these had a cauliflower-like tumor mass on the soft palate, representing co-existing squamous cell carcinoma of the soft palate. The induced nasopharyngeal tumors were all squamous cell carcinomas, most of which were well-differentiated (Fig. 1). The cancer

Table 3. Types and site distribution of tumors induced by DNP

without lymph node metastasis.

the development of cancer.

M, male; F, female

Table 1. NPC induced in rats by DEN and DMBA

The induced tumors resembled human nasopharyngeal cancers but were of a welldifferentiated type, including squamous cell carcinoma (Grade I and II), occasional multiform cell type, and papillary or basal cell carcinomas [57, 72, 73]. The serial sections of nasopharynx enabled us to trace number, sites, and distribution, as well as histogenesis of experimental NPC. This model could be helpful in further study on nasopharyngeal carcinogenesis. The finding that chemical carcinogens such as nitrosamine and aromatic hydrocarbon compounds are capable of inducing nasopharyngeal carcinoma in rats suggests that chemical carcinogens might be one of the etiological factors of human nasopharyngeal carcinoma.

## **3.2.3 Induction of nasopharyngeal carcinoma by nitroso-compounds**

DEN, DNP, and cyclic nitrosocompounds including nitrosomorpholin could induce NPC. The next step was to facilitate studies on pathomorphology, histogenesis, and carcinogenesis of experimental NPC. Two methods were used to minimize the occurrence of hepatoma in DEN-treated rats: the rats were injected intramuscularly with vitamin B12 twice a week and given glucose in their drinking water at the time of DEN administration. DEN was sometimes given through rectal instillation because it causes little damage to this organ. Rats were treated with cyclic nitroso-compounds by two treatment methods, *s.c.* injection and nasopharyngeal instillation. The results of these studies are shown in Table 2 [72, 73].


Inst, instillation; Inj, injection; Adm, administration; Vit, vitamin

Table 2. Induction of NPC by DEN administered by various routes

The induced tumors resembled human nasopharyngeal cancers but were of a welldifferentiated type, including squamous cell carcinoma (Grade I and II), occasional multiform cell type, and papillary or basal cell carcinomas [57, 72, 73]. The serial sections of nasopharynx enabled us to trace number, sites, and distribution, as well as histogenesis of experimental NPC. This model could be helpful in further study on nasopharyngeal carcinogenesis. The finding that chemical carcinogens such as nitrosamine and aromatic hydrocarbon compounds are capable of inducing nasopharyngeal carcinoma in rats suggests that chemical carcinogens might be one of the etiological factors of human

DEN, DNP, and cyclic nitrosocompounds including nitrosomorpholin could induce NPC. The next step was to facilitate studies on pathomorphology, histogenesis, and carcinogenesis of experimental NPC. Two methods were used to minimize the occurrence of hepatoma in DEN-treated rats: the rats were injected intramuscularly with vitamin B12 twice a week and given glucose in their drinking water at the time of DEN administration. DEN was sometimes given through rectal instillation because it causes little damage to this organ. Rats were treated with cyclic nitroso-compounds by two treatment methods, *s.c.* injection and nasopharyngeal instillation. The results of these studies are shown in Table 2 [72, 73].

**3.2.3 Induction of nasopharyngeal carcinoma by nitroso-compounds** 

Inst, instillation; Inj, injection; Adm, administration; Vit, vitamin

Table 2. Induction of NPC by DEN administered by various routes

Table 1. NPC induced in rats by DEN and DMBA

nasopharyngeal carcinoma.

The data showed that DEN instillation resulted in the highest incidence of NPC. The highest incidence of tumor was in the group treated with DEN and vitamin B12, followed by the group given nitrosomorpholin carcinogen. Two tumors with a nodular and ulcerated appearance were visible to the naked eye, one in DEN group and one in nitrosomorpholin group. Both cases were squamous cell carcinomas, partly involving or arising from the soft palate. These tumors invaded surrounding stroma, nerve bundles, and salivary glands without lymph node metastasis.

A few other points were of interest: (a) many squamous carcinomas occurred in neighborhood of auditory pharyngeal tubes or extended along tube walls, indicating that pathological lesions occurred in nasopharyngeal cavity. (b) In nasal cavities of some rats with extensive tumor, the tumor was found only in nasopharyngeal region without invasion of the nasal cavities or the base of the skull. Under naked-eye examination, no tumor was visible in the esophagus or other organs except the liver. (c) Subcutaneous injection of cyclic nitrosamines induced a considerably high incidence of nasopharyngeal tumors, providing some clues to organ specificity of carcinogens. (d) It is worth noting that administration of vitamin B12 markedly increased the incidence of nasopharyngeal cancer (p<0.01), but significantly decreased the incidence of hepatoma. Poirier et al [58] observed that vitamin B12 enhanced hepatic carcinogenesis and shortened the animals' lives. In the present experiments, vitamin B12 increased the incidence of NPC in the DEN group but had the opposite effect in the DNP group. The precise mechanism of this complicated relationship between the carcinogen and nutritional factors needs further study. (e) Vitamin B12, piperazine, and morpholin are usually used in clinics or in pharmaceutical chemistry. Some of them are precursors of the carcinogenic substance and some have an enhancing effect on the development of cancer.

Further investigation into experimental induction of NPC showed that N-nitrosomorpholin and DNP could induce NPC in rats, and subcutaneous injection of DNP could induce NPC without complications of liver cancer. These results are summarized in Table 3 [72, 73].


M, male; F, female

Table 3. Types and site distribution of tumors induced by DNP

A total of 54 rats developed NPC in this experiment. In addition, 39 cancers of the nasal cavities, 32 esophageal tumors, and a few other tumors were observed. Upon postmortem examination, six rats were found to have gross tumor masses in nasopharynx, and two of these had a cauliflower-like tumor mass on the soft palate, representing co-existing squamous cell carcinoma of the soft palate. The induced nasopharyngeal tumors were all squamous cell carcinomas, most of which were well-differentiated (Fig. 1). The cancer

Chemical Carcinogenesis and Nasopharyngeal Carcinoma 69

In the normal rat, cephalic and middle portion of nasopharyngeal cavity is covered by ciliated columnar epithelium, while caudal portion is covered by squamous and columnar

In the soft palates of tumor-bearing rats, nodules ranging from1-2 mm to 1cm in diameter were observed by palpation. Some of the invasive tumors were ulcerative cauliflowerlike or nodular in appearance when the soft palate was cut open, cancerous ulcer with elevated irregular edge was observed when cancerous ulcer with elevated irregular edge was cut open [24]. Under the microscope, cancer cell foci in different stages of differentiation could be found in many serial sections, indicating the multiple growth pattern of malignancy (Fig.1. DNP- induced early nasopharyngeal cancer. Wistar rats were injected subcutaneously with DNP in a stumped needle at a dosage of 40 mg/kg, twice a week and 38 times in total to accumulation dose of 99.5–122 mg per rat. The rats were sacrificed to collect nasopharyngeal samples at 308 days after DNP injections. Nasopharyngeal samples were histopathologically examined under microscope 200). A localized solitary malignancy was observed in rats that received carcinogen for a short period. Tumors often developed at the base of nasopharynx or at the junction of the base and lateral side of nasopharyngeal wall. Carcinomas in situ, and early invasive or infiltrative carcinomas were observed, and

The diagnostic criterion of experimental NPC is generally that of human nasopharyngeal cancer, but the structural characteristics of rat nasopharynx must also be taken into consideration. For example, the epithelium at nasopharynx base, especially at nasopharynx roof next to skull base, consists of two or three layers of cells surrounded by bony plates[59]. The observed malignancy may be composed of basal cells with thin solid strands of cancer

Carcinoma in situ, early invasive carcinoma, and infiltrative carcinoma were observed in tumor-bearing rats. Carcinoma in situ might arise from epithelium of normal thickness, or from highly hyperplastic or atrophic epithelium (Fig.2. DEN- induced nasopharyngeal cancer. Wistar rats were injected subcutaneously with DEN in a stumped needle at a dosage of 40 mg/kg, twice a week and 38 times in total to accumulation dose of 89.8–119.3 mg per rat. The rats were sacrificed to collect nasopharyngeal samples at 365 days after DEN injections. Nasopharyngeal samples were histopathologically examined under microscope). It often originates from columnar epithelium and evolves through squamous cell metaplasia, atypical hyperplasia, and Grade I and II anaplasia. The early invasive lesions were of two types: arising from basal layer with normal looking superficial layers, or extending into stroma (primarily seen in the DEN group). Downward growth of the lesion, which formed trabecular, branching, rounded, or small square nests, infiltrated into the stroma. The neoplasms were usually moderately or even poorly differentiated squamous carcinomas. This downward growth of neoplasms, which often occurs in human NPC, may cause difficulty in early detection or in producing a good smear of exfoliative cells for

**4. Morphologic and histogenetic studies of experimental NPC** 

epithelium.

cells.

cytological diagnosis [57,72,73].

**4.1 Pathology of the experimental NPC** 

were of a well-differentiated squamous cell type [73].

pattern observed in this experiment was somewhat different from that of NPC induced by diethylnitrosamine. The cancer cells invaded the stroma in trabecular or branched cords, but more frequently grew intraepithelially to form patches of masses before invading the stroma. A lymphatic cancer embolus consisting of poorly differentiated cancer cells was found in a case of invasive NPC. No metastatic foci could be found in the lymph nodes of this animal [72, 73].

Fig. 1. DNP- induced early nasopharyngeal cancer **(**Original magnification, ×200**).** Wistar rats were injected subcutaneously with DNP in a stumped needle at a dosage of 40 mg/kg, twice a week and 38 times in total to accumulation dose of 99.5–122 mg per rat. The rats were sacrificed to collect nasopharyngeal samples at 308 days after DNP injections. Nasopharyngeal samples were histopathologically examined under microscope 200.

Most of nasal cavity tumors developed from nasal turbinates. Occasionally, tumors destroyed nasal bone and bulged out as a local prominence. The soft tumor mass with a pink-grey tint frequently destroyed cribriform plate and invaded or replaced olfactory bulb, but it was well demarcated from brain tissue. The tumors of nasal cavity were of different histological types, including squamous cell carcinoma, adenocarcinoma, and the so-called "olfactory neuroepithelial tumor." The olfactory neuroepithelial tumors had a pleomorphic histology that suggested that these tumors originated from epithelia. On gross examination, most of esophageal tumors were multiple papillomas, that developed mainly from the upper and middle parts of esophagus; these papillomas were histologically confirmed as squamous cell carcinoma [73].

Some remaining points need to be clarified: (a) the induced rat NPC was of a squamous cell type. Most of tumors were well-differentiated and developed from base and lateral side of nasopharynx, and some co-existed with squamous carcinoma of soft palate mucosa. There was rarely distant metastasis; in this regard NPC induced by nitroso-compounds was somewhat different from human NPC. (b) DNP-induced NPC and cancer of nasal cavities and esophagus in rats resulted from a subcutanrouic effect. (c) Nitroso-compounds often exist as cis- and trans-isomers, and rapid axial-equatorial conversion of dinitrospiperazin and the respective carcinogenicity of these isomers should be explored in the future.
