An animal model for the rapid induction of tongue neoplasms in human c-Ha- ras proto-oncogene transgenic rats by 4-nitroquinoline 1-oxide: its potential use for preclinical chemoprevention studies

Abstract

Oral squamous cell carcinoma is one of the most common human neoplasms, and prevention of this malignancy requires a better understanding of its carcinogenesis process. To this end, we tried to establish an animal model using the human c-Ha-ras proto-oncogene-carrying transgenic (Tg) rats and the carcinogen 4-nitroquinoline 1-oxide (4-NQO). 4-NQO (20 p.p.m.) was administered to Tg and non-Tg rats for 8 weeks in their drinking water, and then the occurrence of tongue carcinogenesis was compared during the experimental period of 22 weeks. In addition, we determined the DNA ploidy in tongue lesions and examined the immunohistochemical expression of five biomarkers such as cyclin D1, glutathione S-transferase placental form, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and beta-catenin. Next, the cancer chemopreventive effects of nimesulide, pioglitazone and a synthetic geranylated derivative, which have been reported to be inhibitors of tongue carcinogenesis, were examined in Tg rats treated with 4-NQO. Either during or after treatment with 4-NQO in the drinking water, tongue dysplasia and tumors were observed on the tongues of both Tg and non-Tg rats, with a greater incidence and multiplicity in Tg rats. Histopathologically, squamous cell dysplasia, papilloma and carcinoma with or without invasion were present in the tongue. Immunohistochemistry revealed that expression levels against five biomarkers increase with disease progression, and the changes correlated with those of the DNA ploidy pattern. Interestingly, a strong expression of COX-2, iNOS and beta-catenin was observed on the invasive front of squamous cell carcinomas. A subsequent chemoprevention study using Tg rats showed that the chemicals tested suppressed the occurrence of tongue carcinomas when they were administered after 4-NQO-exposure. These results may thus indicate that our 4-NQO-induced Tg rat tongue carcinogenesis model simulates many aspects of human oral carcinogenesis and it can be applied for an analysis of oral cancer development while also helping to identify potentially effective cancer chemopreventive agents against oral cancer.

Enhancement of tongue carcinogenesis in Hras128 transgenic rats treated with 4-nitroquinoline 1-oxide

“…These results clearly show that Hras128 rats are more sensitive to 4NQO induced tongue carcinogenesis than SD rats. This susceptibility to a carcinogen is in accordance with previous experiments demonstrating that Hras128 rats are more sensitive to a 7,12-dimethylbenz[a]anthracene (DMBA) or 4NQO in the mammary or tongue tumors than were wild-type SD rats (11,21). In addition, Miyamoto et al demonstrated that the rasH2 mouse carcinogenesis model is highly susceptible to 4NQO induced tongue and esophageal tumors (12).…”

“…Human oral cancer is a focal disorder (3), and experimental studies have indicated that there is a multistage process in the development of the oral lesions, in which dysplasias process to SCC through PAP (11,12). The results obtained in the present study showing that Hras128 and SD rats developed DH and tumors in a time-dependent manner (Fig.…”

“…The incidence and number of tongue tumors (PAP and SCC) also markedly increased after 6 weeks of 4NQO exposure, and this increase persisted until the end of the experiment (at experimental-week 14), when compared with these lesions in SD rats. In conventional 4NQO initiated carcinogenesis models, oral tumors are induced during 22-32 experimental weeks (11,22). In the present model, tongue tumors can be induced in only a short period of 14 experimental weeks.…”

“…The 4NQO-induced oral cancer is derived from papilloma through hyperplastic epithelium and dysplasia (11), and this multistage process mimics the development of these malignancies in human (11,12). Recent reports by Tsuda and his colleagues demonstrate that transgenic rat carrying human c-Ha-ras proto-oncogene, termed Hras128 rat [Jcl/SD-TgN(HrasGen)128Ncc], is highly susceptible to tumor induction in various organ sites including mammary gland, esophagus, skin, urinary bladder and tongue (13)(14)(15).…”

Whole slide image cytometry: a novel method to detect abnormal DNA content in Barrett's esophagus

“…This has been reported previously and attributed to the intrinsic characteristics of performing DNA ploidy analysis using thin tissue sections. 43,64 Thick tissue sections (e.g., 410 μm) create difficulty in nuclei segmentation, especially in situations of touching and overlapping nuclei. Therefore, biased sampling error could happen where smaller and isolated nuclei are preferred.…”