**2. Carcinogens related to the etiology of human NPC**

NPC occurs most frequently in Southeast Asia and Africa. The highest incidence rate is reported to be in the southern provinces of China, and NPC contributes to a high mortality rate among Chinese people [31]. There are many articles and publications focusing on the viral and hereditary factors associated with NPC but few on chemical factors such as environmental carcinogens. The importance of each factor may vary between different tumors and in different areas of the world [32]. Moreover, it appears that a multiple-factor concept of cancer etiology may be relevant to human NPC and chemical carcinogens should be taken into consideration within this context. Although numerous chemical agents are suspected to be related to human tumors, the discussion here will be limited to certain polycyclic hydrocarbons, nitrosamines, and some related compounds that might play a more intimate role in the etiology of human NPC [33].

#### **2.1 Polycyclic aromatic hydrocarbons**

Since Pott's [34] observation on scrotal cancer and the first demonstration of the induction of cancer in animals by painting coal tar on the skin, the importance of hydrocarbons in human carcinogenesis has been extended to include a possible role as a risk factor in human NPC.

Some clinicians have paid attention to hydrocarbon as one of the etiological factors of human NPC due to the continued exposure of their patients to coal dust. Furthermore, Schoental et al [35] reported that the incidence rate of NPC was higher among the inhabitants of the mountainous district than in those living at low altitude in the flatlands. It was subsequently found that the inhabitants of districts with a higher incidence of NPC warmed themselves by burning firewood. In low and poorly ventilated living rooms, the accumulation of sooty fumes results in environmental pollution. In the high incidence region, studies found that the benzo(a)pyrene concentration reached a level of 85-29 µg/1,000 m3 , and that of benzanthrene reached 79-515 µg/1,000 m3, so it was believed that NPC in this district is related to exposure to polycyclic aromatic hydrocarbons [36].

Fong YY et al [37] tried to instill the oil extraction of soot obtained from the houses of NPC patients into the nasal cavities of the mice three times a week, but this did not induce NPC. The control group was treated with methylcholanthrene and developed nasal cavity tumors but not NPC. He concluded that nasopharyngeal mucosa is not sensitive to chemical carcinogens. Similarly, Lo et al [38] injected carcinogenic agents methylcholanthrene and benzo(a)pyrene into the nasopharyngeal region of rabbits, rats, mice, and dogs, and did not observe any positive results. In contrast, Pan et al [72, 73] inserted DMBA crystals into ectopically implanted nasopharyngeal tissues of homologous mice, and two cases of squamous carcinoma were induced in 20 mice. This result implied that nasopharyngeal epithelium could be induced to develop carcinoma, and was not insusceptible to these carcinogens.

Toth B et al [39] injected benzo(a)pyrene and dimethylbenzanthrere into AKR mice through the posterior nasal orifice. The induced tumors were mainly located at the hard palate and nasal cavity. Pan et al [72] developed a method for inducing NPC in rats. Long thin polyethylene tubes loaded with benzo(a)pyrene, DMBA, or 3-methylcholanthrene (MC) were inserted into the nasopharyngeal cavities of rats under anesthesia, resulting in squamous carcinoma of the nasopharynx. One animal developed a cancerous ulcer on the mucosa of the nasopharynx. This nasopharynx cancer was a grade III squamous cell carcinoma, which malignant cells grew upwards protruding into the cavity or downwards into the stroma. The incidence rates of the respective groups were as high as those in the group of rats treated with DMBA or DEN.

#### **2.2 Nitrosamines**

62 Carcinogenesis, Diagnosis, and Molecular Targeted Treatment for Nasopharyngeal Carcinoma

0.028 to 4.54 mg/kg [25] and are converted into carcinogenic N-nitrosocompounds including N,N'-dinitrosopiperazine after food intake [26]. N-nitrosodimethylamine is the predominant volatile nitrosamine in salted fish. In addition, some bacteria can also induce conversion of nitrate to nitrite, which forms important carcinogenic N-nitroso compounds [26]. Experiments in rats have demonstrated the carcinogenicity of nitrosamines and Nnitroso compounds such as diethylnitrosamine (DEN), dimethlbenzanthracene anthracene

NPC occurs most frequently in Southeast Asia and Africa. The highest incidence rate is reported to be in the southern provinces of China, and NPC contributes to a high mortality rate among Chinese people [31]. There are many articles and publications focusing on the viral and hereditary factors associated with NPC but few on chemical factors such as environmental carcinogens. The importance of each factor may vary between different tumors and in different areas of the world [32]. Moreover, it appears that a multiple-factor concept of cancer etiology may be relevant to human NPC and chemical carcinogens should be taken into consideration within this context. Although numerous chemical agents are suspected to be related to human tumors, the discussion here will be limited to certain polycyclic hydrocarbons, nitrosamines, and some related compounds that might play a

Since Pott's [34] observation on scrotal cancer and the first demonstration of the induction of cancer in animals by painting coal tar on the skin, the importance of hydrocarbons in human carcinogenesis has been extended to include a possible role as a risk factor in human NPC. Some clinicians have paid attention to hydrocarbon as one of the etiological factors of human NPC due to the continued exposure of their patients to coal dust. Furthermore, Schoental et al [35] reported that the incidence rate of NPC was higher among the inhabitants of the mountainous district than in those living at low altitude in the flatlands. It was subsequently found that the inhabitants of districts with a higher incidence of NPC warmed themselves by burning firewood. In low and poorly ventilated living rooms, the accumulation of sooty fumes results in environmental pollution. In the high incidence region, studies found that the benzo(a)pyrene concentration reached a level of 85-29 µg/1,000 m3 , and that of benzanthrene reached 79-515 µg/1,000 m3, so it was believed that

NPC in this district is related to exposure to polycyclic aromatic hydrocarbons [36].

Fong YY et al [37] tried to instill the oil extraction of soot obtained from the houses of NPC patients into the nasal cavities of the mice three times a week, but this did not induce NPC. The control group was treated with methylcholanthrene and developed nasal cavity tumors but not NPC. He concluded that nasopharyngeal mucosa is not sensitive to chemical carcinogens. Similarly, Lo et al [38] injected carcinogenic agents methylcholanthrene and benzo(a)pyrene into the nasopharyngeal region of rabbits, rats, mice, and dogs, and did not observe any positive results. In contrast, Pan et al [72, 73] inserted DMBA crystals into ectopically implanted nasopharyngeal tissues of homologous mice, and two cases of squamous carcinoma were induced in 20 mice. This result implied that nasopharyngeal

(DMBA), and Dinitrosopiperazine (DNP) [6, 15, 30].

more intimate role in the etiology of human NPC [33].

**2.1 Polycyclic aromatic hydrocarbons** 

**2. Carcinogens related to the etiology of human NPC** 

Since Magee [40] first described the toxicity and carcinogenicity of dimethylnitrosamine in rats, the carcinogenicity of nitroso-compounds in different animals has aroused increasing interest and received intensive investigation. A series of reports confirmed that nitrosocompounds could induce a variety of malignant tumors in a great number of different animal species [41-44]. Moreover, nitroso-compounds may result from the interaction of nitrites and secondary amines. These precursors are produced from nitrosamines by bacterial action in the acidic environment of stomach or alkaline intestinal contents [45, 46]. The putative role of nitroso-compounds in the induction of NPC has fascinated many researchers. Ho [47] raised the tentative assumption that the high incidence of NPC in Hong Kong was due to the ingestion of salted fish as main protein source. An appreciable amount of dimethylnitrosamine was reportedly detected in salted fish in the markets of Hong Kong [48]. However, a control survey carried out in Guangzhou demonstrated no such relationship between NPC and the intake of salted fish [4].

Generally, the saliva of NPC patients has a higher nitrite content and lower nitrate content than that of normal individuals. In addition, the urinary nitrite content of NPC patients was higher than those of normal controls. These differences were statistically significant in Sihui County [49]. It was suggested that nitrate content of saliva from NPC patients might be reduced by microorganisms in oral cavity; however, the precise mechanism needs to be further investigated. Yi Z et al [50] analyzed nitrate levels in saliva and urine samples collected from 75 NPC patients. The nitrate content in the urine sample of these patients was considerably lower than that of normal subjects, whereas the nitrite content was significantly higher in the urine samples of NPC patients. It was proposed that NPC patients might possess certain reduction mechanisms that could reduce nitrates into nitrites, thus resulting in increased urinary excretion of nitrites and enhanced endogenous synthesis of nitroso-compounds.

There were no reports on experimental nasopharyngeal carcinoma induced by nitroso compounds as carcinogens until 1972. Huang et al [24] reported that out of 22 white rats fed with salted fish, four developed nasal tumors but none developed nasopharyngeal cancer. However, in 1972, Pan et al [72, 73] successfully induced NPC in rats using nitrosocompounds, thus providing new clues in the investigation of the cause of NPC.

Chemical Carcinogenesis and Nasopharyngeal Carcinoma 65

is poorly differentiated. Stratified squamous epithelium appears 10 days after birth. The

Generally, potent carcinogen with short induction time is chosen as tumor-inducing agent, for example, benzo(a)pyrene for lung cancer induction [54] and aflatoxin for liver cancer [55]. Human NPC is too complicated to determine one or two factors because there are many factors involved in NPC development, such as viral and genetic factors, chemical carcinogens in the environment [56]. Moreover, the etiology of human cancers can be complicated, and might not be attributed to a single factor or initiation event [2]. The combined action of chemical carcinogens, viruses, and genetic factor, and also co-

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

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

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

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.

nasopharyngeal epithelium of 60-day-old rats is similar to that of adult rats.

**3.1.2 Selection of carcinogens and induction methods** 

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

**3.2.1 Establishment of the NPC animal model** 

carcinogens should be discussed.

discussed below.

these chemical compounds.

**DMBA** 
