Investigation of in vivo unscheduled DNA synthesis in rabbit corneas following instillation of genotoxic agents.

PURPOSE: An unscheduled DNA synthesis (UDS) test is used for in vitro or in vivo genotoxicity evaluation. The UDS test with hepatocytes is well established; however, drug exposure levels at the application site for topically administered drugs (e.g. ophthalmic drugs) often exceed the exposure levels for systemic administration. To establish in vivo genotoxicity on the ocular surface, we performed the UDS test using rabbit corneas from eyes subjected to instillation of genotoxic agents. MATERIALS AND METHODS: Five genotoxic agents - 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat); acridine orange; ethidium bromide; acrylamide; and 4-nitroquinoline 1-oxide (4-NQO) - were instilled once onto both eyes of male Japanese white rabbits. Physiological saline or a general vehicle for ophthalmic solution were instilled as the negative controls. Dimethyl sulfoxide was instilled as the vehicle control. Isolated corneas were incubated with tritium-labelled thymidine and the number of sparsely labelled cells (SLCs, cells undergoing UDS) was counted by autoradiography. RESULTS: Statistically significant increases in the mean appearance rates of SLCs in the corneal epithelium were noted in paraquat-, acridine orange-, ethidium bromide-, and 4-NQO-treated eyes compared with those of the controls. These increases generally appeared in a dose-dependent manner. Acrylamide did not induce an increase in the mean appearance rates of SLCs, presumably because it caused the generation of fewer metabolites in the cornea. CONCLUSIONS: UDS tests revealed DNA damage in the cornea epitheliums treated with well-known genotoxic agents. These results suggest that the UDS test is one of the useful tools for the assessment of in vivo genotoxicity on the ocular surface in the development of ophthalmic drugs.

Beta-adrenergic blocker inhibits oral carcinogenesis and reduces tumor invasion.

PURPOSE: Beta-adrenergic signaling can influence cancer progression and the use of beta blockers as adjuvant drugs in oncologic patients has been suggested. However, the involvement of beta-adrenergic blockers in tumorigenesis is poorly understood. This study investigated the action of beta-adrenergic blocker propranolol on tumor onset using a preclinical model of chemically induced oral cancer. METHODS: Thirty-two male Wistar rats were subjected to daily subcutaneous injection of beta-blocker propranolol (10 mg/kg; SubQ), while another 32 rats received only a PBS injection (sham group). One week after starting propranolol treatment, all rats were submitted to chemical induction of oral carcinogenesis with 4-nitroquinoline-1-oxide (4NQO). After 16 weeks, they were assessed for occurrence of oral squamous cell carcinoma (OSCC), in addition to measurement of tumor volume and thickness, and tissue levels of cytokines IL-6, TNF-alpha and IL-10 in the tumor microenvironment. RESULTS: Propranolol treatment reduced the occurrence of OSCC by 31%, 95% CI ( - 127, 216). Beta-adrenergic blocker significantly decreased thickness of OSCC when compared with PBS. Rats treated with propranolol exhibited a lower tumor volume when compared with control rats, but this result did not reach statistical significance. Tumors from propranolol-treated rats exhibited reduced concentrations of pro-inflammatory cytokines IL-6 and TNF-α. There was no difference in the IL-10 levels between tumors from propranolol- and sham-treated rats. CONCLUSION: Beta-adrenergic signaling may be one of the mechanisms associated with chemically induced oral carcinogenesis.

An Infection-Based Murine Model for Papillomavirus-Associated Head and Neck Cancer.

Human papillomavirus (HPV) is the most common sexually transmitted pathogen, and high-risk HPVs contribute to 5% of human cancers, including 25% of head and neck squamous cell carcinomas (HNSCCs). Despite the significant role played by HPVs in HNSCC, there is currently no available in vivo system to model the process from papillomavirus infection to virus-induced HNSCC. In this paper, we describe an infection-based HNSCC model, utilizing a mouse papillomavirus (MmuPV1), which naturally infects laboratory mice. Infections of the tongue epithelium of two immunodeficient strains with MmuPV1 caused high-grade squamous dysplasia with early signs of invasive carcinoma over the course of 4 months. When combined with the oral carcinogen 4-nitroquinoline-1-oxide (4NQO), MmuPV1 caused invasive squamous cell carcinoma (SCC) on the tongue of both immunodeficient and immunocompetent mice. These tumors expressed markers of papillomavirus infection and HPV-associated carcinogenesis. This novel preclinical model provides a valuable new means to study how natural papillomavirus infections contribute to HNSCC.IMPORTANCE The species specificity of papillomavirus has limited the development of an infection-based animal model to study HPV-associated head and neck carcinogenesis. Our study presents a novel in vivo model using the mouse papillomavirus MmuPV1 to study papillomavirus-associated head and neck cancer. In our model, MmuPV1 infects and causes lesions in both immunodeficient and genetically immunocompetent strains of mice. These virally induced lesions carry features associated with both HPV infections and HPV-associated carcinogenesis. Combined with previously identified cancer cofactors, MmuPV1 causes invasive squamous cell carcinomas in mice. This model provides opportunities for basic and translational studies of papillomavirus infection-based head and neck disease.

Role of Electron Microscopy in Early Detection of Altered Epithelium During Experimental Oral Carcinogenesis.

Early detection of altered epithelium can help in controlling the further progression by timely intervention. Alterations in cellular adhesion are one of the hallmarks of cancer progression, which can be detected at the intracellular level using high-resolution electron microscopy. This study aimed to evaluate the role of electron microscopy in the establishment of ultrastructural markers for early detection of altered epithelium using tissues from 4-Nitroquinoline-1-Oxide (4NQO) induced rat tongue carcinogenesis. Our previous study using light microscopy displayed no histopathological alterations in 4NQO treated tissues until 40 days of treatment, while dysplasia, papilloma and carcinoma were detected at 80/120, 160 and 200 days, respectively. However, electron microscopy detected alterations such as detachment of desmosomes from cell membranes and their clustering in the cytoplasm, increased tonofilaments, keratohyaline granules and thickened corneum in 40 days treated corresponding tissues. These alterations are apparent with hyperkeratosis/hyperplasia but remained undetected using light microscopy. Further, in dysplasia, papilloma and carcinoma, gradual and significant loss of desmosomes, leading to the significant widening of intercellular spaces, was observed using iTEM software. These parameters may serve as indicators for progression of oral cancer. Our results highlight the importance of electron microscopy in the early detection of subcellular changes in the altered epithelium.

Modulation of IL-1ß reprogrammes the tumor microenvironment to interrupt oral carcinogenesis.

Head and neck squamous cell carcinoma (HNSCC) development is a multistage process includes the normal, dysplasia and squamous cell carcinoma (SCC) stages. Recently, increasing evidence has suggested that the tumor microenvironment (TME) is an integral part of malignant transformation. Exploring certain key node genes in TME for future intervention in dysplasia to interrupt oral carcinogenesis was the primary goal of this research. To achieve this goal, systems biology approaches were first applied to the epithelia and fibroblasts collected at sequential stages in a 4-nitroquinoline-1-oxide (4NQO) -induced rat oral carcinogenesis model. Through bioinformatics network construction, IL-1ß was identified as one of the key node genes in TME during carcinogenesis. Immunohistochemical staining of human and rat samples demonstrated that IL-1ß expression patterns were parallel to the stages of malignant transformation. Silencing IL-1ß with lentivirus-delivered shRNA significantly inhibited oral squamous cell carcinoma cell growth both in vivo and in vitro. Based on these findings, we hypothesized that IL-1ß may be a chemoprevention target in TME during oral carcinogenesis. Therefore, we targeted IL-1 in the TME by oral mucosal injection of an IL-1 receptor antagonist in 4NQO rats. The results demonstrated that targeting IL-1 could interrupt oral carcinogenesis by reprogramming the TME.

Comparison of integrated genotoxicity endpoints in rats after acute and subchronic oral doses of 4-nitroquinoline-1-oxide.

During interlaboratory validation trials for the Pig-a gene mutation assay we assessed the genotoxicity of 4-nitroquinoline-1-oxide (4NQO) across endpoints in multiple tissues: induction of Pig-a mutant red blood cells (RBCs) and reticulocytes (RETs); micronucleated RETs (MN RETs); and DNA damage in blood and liver via the alkaline Comet assay (%tail intensity [TI]). In a previous subchronic toxicity study with 28 daily doses, biologically meaningful increases were observed only for Pig-a mutant RBCs/RETs while marginal increases in the frequency of MN RET were observed, and other clastogenic endpoints were negative. Follow up acute studies were performed using the same cumulative doses (0, 35, 70, 105, and 140 mg/kg) administered in a bolus, or split over three equal daily doses, with samples collected up to 1 month after the last dose. Both of the acute dosing regimens produced similar results, in that endpoints were either positive or negative, regardless of 1 or 3 daily doses, but the three consecutive daily dose regimen yielded more potent responses in TI (in liver and blood) and Pig-a mutant frequencies. In these acute studies the same cumulative doses of 4NQO induced positive responses in clastogenic endpoints that were negative or inconclusive using a subchronic study design. Additionally, a positive control group using combination doses of cyclophosphamide and ethyl methanesulfonate was employed to assess assay validity and potentially identify a future positive control treatment for integrated genetic toxicity studies.

Suppression of Rat Oral Carcinogenesis by Agonists of Peroxisome Proliferator Activated Receptor γ.

Peroxisome-proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor that regulates cell proliferation, differentiation, and apoptosis. In vivo studies were performed to evaluate the activities of two thiazolidinedione PPARγ agonists, rosiglitazone and pioglitazone, as inhibitors of oral carcinogenesis in rats. Oral squamous cell carcinomas (OSCC) were induced in male F344 rats by 4-nitroquinoline-1-oxide (NQO; 20 ppm in the drinking water for 10 weeks). In each study, groups of 30 NQO-treated rats were exposed to a PPARγ agonist beginning at week 10 (one day after completion of NQO administration) or at week 17 (7 weeks post-NQO); chemopreventive agent exposure was continued until study termination at week 22 (rosiglitazone study) or week 24 (pioglitazone study). Administration of rosiglitazone (800 mg/kg diet) beginning at week 10 increased survival, reduced oral cancer incidence, and reduced oral cancer invasion score in comparison to dietary controls; however, chemopreventive activity was largely lost when rosiglitazone administration was delayed until week 17. Administration of pioglitazone (500 mg/kg diet beginning at week 10 or 1000 mg/kg diet beginning at week 17) induced significant reductions in oral cancer incidence without significant effects on OSCC invasion scores. Transcript levels of PPARγ and its three transcriptional variants (PPARγv1, PPARγv2, and PPARγv3) were not significantly different in OSCC versus age- and site-matched phenotypically normal oral tissues from rats treated with NQO. These data suggest that PPARγ provides a useful molecular target for oral cancer chemoprevention, and that overexpression of PPARγ at the transcriptional level in neoplastic lesions is not essential for chemopreventive efficacy.

The in vitro PIG-A gene mutation assay: mutagenicity testing via flow cytometry based on the glycosylphosphatidylinositol (GPI) status of TK6 cells.

The X-linked PIG-A gene is involved in the biosynthesis of the cell surface anchor GPI, and its inactivation may serve as a new marker for mutagenicity. The in vivo PIG-A gene mutation assay is currently being validated by several groups. In this study, we established a corresponding in vitro variant of the PIG-A assay applying B-lymphoblastoid TK6 cells. PE-conjugated antibodies against the GPI-anchored proteins CD55 and CD59 were used to determine the GPI status via multicolor flow cytometry. Mutant spiked TK6 cell samples were analyzed, and mutants were quantified with even small numbers being quantitatively recovered. To validate our approach, mutant spiked cell samples were analyzed by flow cytometry and proaerolysin selection in parallel, yielding a high correlation. Further, we developed a procedure to reduce the background level of preexisting mutant cells to lower than 20 in 10(6) cells to increase the sensitivity of the assay. Spontaneous rate of GPI deficiency was investigated being 0.76 × 10(-6)/cell/generation for TK6 cells. The optimal phenotype expression time after ethyl methanesulfonate treatment was found to be 10 days. We applied the in vitro PIG-A assay to demonstrate the mutagenicity of ethyl methanesulfonate, 4-nitroquinoline 1-oxide and UV-C irradiation in a dose-dependent and statistically significant manner. Pyridine and cycloheximide were included as negative controls providing negative test results up to 10 mM. These data suggest that the in vitro PIG-A assay could complement the in vivo PIG-A assay with some distinct advantages compared to other in vitro mammalian mutagenicity tests.

FDG-PET/CT of head and neck squamous cell carcinoma in a rat model.

PURPOSE: The aim of this study was to demonstrate the ability to use human clinical positron emission tomography/computed tomography (PET/CT) to detect and investigate head and neck cancers chemically induced by 4-nitroquinoline-1-oxide (4-NQO) in a rat model. STUDY DESIGN: The study design was prospective animal research. PROCEDURES: A head and neck squamous cell carcinoma was established in 20 immunocompetent rats, who drank a 4-NQO solution during 16 weeks. 2-Deoxy-2-[F-18]fluoro-D: -glucose (FDG)-PET/CT was performed for five of them, 34 weeks after the start of the experiment to characterize the tumors. A day following the FDG-PET/CT, rats were euthanized and pathological features were evaluated by hematoxylin-eosin staining. RESULTS: All rats had head and neck tumor at various locations at 34 weeks. Among the five rats selected for having FDG-PET/CT, the clinical examination detected exophytic tumors grown in the oral cavity for three of them (one on the inferior lip, one on the hard palate, and one on the internal side of the cheek). FDG-PET/CT confirmed the presence of those tumors and detected ones located on the base of tongue for three of them. Tumor extensions were characterized and tumor metabolic volumes were measured. The smallest lesion detected measured 3 x 3 x 4 mm. Pathologic examination using hematoxylin-eosin staining confirmed squamous cell carcinoma. CONCLUSIONS: This study demonstrated that FDG-PET/CT is a feasible examination to detect occult primary tumors in rat models. It is useful to follow tumor progression and evaluate therapeutics efficacy.

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.

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.

Genetic predisposition to 4NQO-induced tongue carcinogenesis in the rat.

OBJECTIVE: This study aims to elucidate the genetic basis of predisposition to 4-nitroquinoline 1-oxide (4NQO)-induced tongue cancers (TCs). MATERIALS AND METHODS: We have reported that inbred Dark-Agouti (DA) strain rats were highly susceptible to 4NQO-induced TCs, whereas Wistar/Furth (WF) rats were resistant to tongue squamous cell carcinomas induced by oral administration of 4NQO. Using size and number of the tumours as quantitative parameters, responsible host loci were analysed by an interval mapping of F2 intercross of DA and WF given carcinogenic regimen. Also, loss of heterozygosity (LOH) at these loci was analysed in tongue cancers in (DA x WF) F1. RESULTS: We identified and mapped 5 significant quantitative trait loci (QTL), the Tongue squamous cell carcinoma 1-5 (Tscc1-5), and several other suggestive QTL that determine susceptibility to 4NQO-induced TC. Study of TCs induced in (DA x WF)F1 rats revealed a high frequency of LOH in the chromosomal regions of Tscc2, 3, and 4 and also of suggestive QTL on chromosomes 5 and 6. The fact that LOH was found only in larger TCs indicates that LOH occurred in the process of tumour progression. In most LOH, the allele of the resistant WF strain was lost, suggesting that these loci may encode tumour suppressor genes. In larger TCs, in addition to LOH, point mutations and the methylation of possible candidate genes were accumulated. CONCLUSION: These observations indicate that the 4NQO-induced TC in the rat is a multifactorial disease of a polygenic trait. This model will be useful to understand the complicated genetic basis of predisposition to oral cancers.

PCNA in palate and tongue mucosal dysplastic lesions induced by topically applied 4NQO in desalivated rat.

OBJECTIVES: Saliva has been suggested to have a protective role against carcinogens in the oral cavity in animals. Water-soluble 4NQO is a suitable carcinogen to use in examining the role of saliva in oral cancer. The purpose of this study was to follow the changes induced by the carcinogen at the molecular level, as well as the effect of lack of saliva on these changes. MATERIALS AND METHODS: Topical application to the palate of a 0.5% 4NQO solution dissolved in glycol was used for 4 months to induce malignant transformation in a desalivated rat model. Histomorphometric analysis of proliferating cell nuclear antigen (PCNA), a cell cycle regulator and a proliferation marker, was performed. RESULTS: Manifestation of PCNA significantly increased as the observed histologic changes progressed from hyperkeratosis, to mild or moderate dysplasia, severe dysplasia and squamous cell carcinoma. Differences in manifestation of PCNA among the diagnostic groups was significant (p< 0.05). In the desalivated group, PCNA expression was significantly higher than in control and normal groups, in both tongue and palate after 2 and 4 months (p< 0.05). CONCLUSIONS: An unknown component of saliva has a temporary anti-carcinogenic protective effect, which can both delay and decrease the level of proliferation induced by the carcinogen 4NQO. The specific salivary component and the mechanism by which this protective effect is rendered are yet to be discovered.

Mutagenic activity of 4-nitroquinoline-N-oxide in upper aerodigestive tissue in lacZ mice (MutaMouse) and the effects of 1, 4-phenylenebis(methylene)selenocyanate.

4-Nitroquinoline-N-oxide (4-NQO) was administered to lacZ mice at a concentration of 20 microg/ml in drinking water for 2 weeks, and the mutagenic fractions in a number of organs were assayed. The mutant fractions in tongue, esophagus and other pooled oral tissues were, respectively, 117+/-26, 73+/-15, and 48+/-15 mutants/10(5) plaque-forming units (pfu) (ca. 15-40xbackground). 4-NQO was not mutagenic in lung, liver or colon at conditions used here. We had previously demonstrated that the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC), an established chemopreventive agent, greatly reduced carcinogenicity in 4-NQO in rat tongue, and we observed here that administration of p-XSC (10 ppm se) in the diet for 6 weeks (2 weeks before, during, and 2 weeks after 4-NQO) resulted in a 33% decrease in mutagenesis in oral tissue, a 17% decrease in esophagus, and a slight increase in tongue. Only the decrease in oral tissue reached statistical significance (p<0.04). The results reported here demonstrate that 4-NQO was extremely mutagenic in lacZ mouse tongue, with lower, but highly significant activities in esophagus and other pooled oral tissues. The high activity of 4-NQO in lacZ mouse tongue is consistent with the organ specificity of 4-NQO in the rat. Inhibition of 4-NQO-induced mutagenesis by p-XSC was observed mainly in pooled oral tissues, other than tongue. Possible reasons for the difference between inhibition of mutagenesis and carcinogenesis in tongue are discussed, as well as advantages and disadvantages of in vivo mutagenesis assays as surrogates for carcinogenicity assays in chemoprevention studies.

[A rat model of tongue mucosa squamous cell carcinoma induced by oral administration of 4NQO in drinking water].

OBJECTIVE: To establish a more realistic animal model for oral carcinogenesis which reveals histological and immunological characteristics similar to the human counterpart. METHODS: 0.002% 4-nitroquinoline 1-oxide (4NQO) in drinking water was administered orally to SD rats for 9-32 weeks. Then the rats were killed and their tongues were removed for histological assessment. RESULTS: Gross changes included leukoplakia, erosion, ulcer and papillary appearance on the dorsum of the posterior tongue were present during carcinogenesis. Their corresponding histopathological findings ranged from hyperplasia (HP), mild-moderate dysplasia (mmDP), severe dysplasia (sDP) and in situ carcinoma (ISC) to well-differentiated invasive squamous cell carcinoma (SCC). The severity of lesions corresponded to the duration of administration. The tongues in rats treated with 4NQO for 9 weeks showed HP (80.0%) and mmDP (20.0%); those for 13 weeks showed mmDP (66.6%) and sDP (33.3%); those for 16 weeks showed sDP (55.5%) and ISC (44.4%); and those for 32 weeks showed sDP (12.5%), ISC (12.5%) and SCC (75.0%). The incidence of tongue cancer in rats treated with 4NQO for 9 weeks, 13 weeks, 16 weeks and then observed for 32 weeks was 50.0%, 62.5%, and 77.8%, respectively. No metastases were found. CONCLUSION: 4NQO reliably produced preneoplastic and neoplastic tongue mucosa lesions, which morphologically and histologically mimic human oral carcinogenesis. The rat tongue, a target organ of 4NQO, is not an immunologically privileged site like the hamster buccal pouch. Thus, the model should be appropriate to study molecular mechanism of neoplastic transformation and to assess new treatment modalities of premalignant and malignant lesions of the human oral cavity.

Mutagen sensitivity exhibits a dose-response relationship in case-control studies.

We have been quantitating, as a marker of cancer susceptibility, induced chromatid breaks in lymphocyte cultures exposed to chemical mutagens. This report highlights the consistency of the results from two case-control studies, using different methods of presenting the data. In both the lung cancer case-control study, which used bleomycin, a radiomimetic agent, as the test mutagen, and the melanoma study, which used 4-nitroquinoline-oxide, an UV-mimetic agent, the mean number of breaks/cell was significantly higher in the cases compared with the controls. When the data were dichotomized at the 75th percentile of breaks in the control populations, significantly elevated adjusted odds ratios (3.7 and 5.0, respectively) were detected. Dose-response relationships were evident in both studies when the data were categorized by quartiles of breaks/cell in the controls, with highest risk estimates being in the top quartile of induced breaks. The potential for extending this assay to other cancer sites, using a variety of test mutagens, is exciting.

Proteoglycan changes in carcinogen (4NQO)-treated rat tongue mucosa.

The purpose of this study was to undertake preliminary analyses of the extracellular proteoglycans in carcinogen [4-nitroquinoline N-oxide (4NQO)]-treated rat tongue mucosa. Experimental rats were exposed to twice-weekly applications of 4NQO in propylene glycol for six months, after which the animals were killed. Control and 4NQO-treated tissues were subjected to sequential aqueous extractions of proteoglycans under associative and dissociative conditions, followed by alkaline cleavage of protein-glycosaminoglycan linkages to yield a glycosaminoglycan residue. Tissues subjected to 4NQO applications contained smaller proportions of proteoglycans which were readily soluble under associative and dissociative conditions. Proportionately more proteoglycan remained strongly associated with other intercellular tissue components, being released only by alkaline cleavage. These biochemical alterations in preinvasive 4NQO-treated epithelium and connective tissues, together with an observed associated change in water retention by the connective tissue, occurred prior to actual neoplastic invasion and suggest differences in macromolecular conformation and orderliness. We hypothesize that these changes are related to the phenomenon of neoplastic epithelial invasion.

[Measurement of prostaglandin E2 content in lung tissue of mouse].

A radioimmunoassay (RIA) was applied for the determination of PGE2 levels in the lung from 4 groups of male ICR mice 1) fed a standard diet, 2) fed a 20% corn oil-supplemented diet (HFD) for 2 weeks, 3) given a single s.c. injection of 15 mg/kg of 4NQO, a potent lung carcinogen and 4) given a s.c. injection of 4NQO and subsequently fed HFD for 2 weeks. Animals were sacrificed at week 3, and the lung was carefully excised and frozen in liquid nitrogen to prevent the postmortem synthesis of PGE2. PGE2, extracted from the lung tissue, was purified and then measured with or without adding a known amount of PGE2. The lung levels of PGE2 were shown to be significantly higher in the groups treated with HFD and/or 4NQO than the group fed a standard diet. These results show that the modified RIA method can be used for the measurement of PGE2 contents in the tissue of animals.

Induction of squamous metaplasia, dysplasia and carcinoma in situ of the mouse tracheal mucosa by inhalation of sodium chloride mist following subcutaneous injection of 4-nitroquinoline 1-oxide.

Squamous metaplasia, dysplasia and carcinoma in situ (CIS) were induced in the ICR/Jcl mouse tracheal mucosa by exposure to a mist of 5% NaCl solution following single subcutaneous injection of 4-nitroquinoline 1-oxide (4-NQO). Either subcutaneous injection of 4-NQO or NaCl inhalation alone did not cause any marked change in the tracheal mucosa. The NaCl inhalation is considered to have promotion-like action on the mouse tracheal mucosa after administration of 4-NQO injection.

Initiation and promotion of experimental oral mucosal carcinogenesis in mice.

While there is considerable evidence that skin carcinogenesis proceeds as a step-wise series of changes, little evidence is available to indicate that a similar mechanism applies to oral mucosal carcinogenesis. In the current study, a mouse model of mucosal carcinogenesis was used to examine the effects of repeated applications of the phorbol ester phorbol-12,13-didecanoate (PDD) following various periods of treatment with the carcinogen 4-nitroquinoline-1-oxide (4NQO). All animals were histologically examined at 50 weeks. Oral epithelial dysplasia was seen in animals treated with 4NQO for as little as 2 weeks, and oral squamous cell carcinomas developed in all animals treated with 4NQO for 16 weeks. In those mice treated with PDD as well, carcinomas developed in mice receiving as little as 2 weeks treatment with 4NQO, and 100% of mice treated with 4NQO for 12 weeks, followed by PDD treatment, developed carcinomas. The results indicate that irreversible changes in the oral mucosa of mice occur relatively early during treatment with 4NQO, and the development of carcinomas can be enhanced with subsequent PDD treatment, giving an indication of staged carcinogenesis in this model system.

Experimental tongue carcinoma of rats induced by oral administration of 4-nitroquinoline 1-oxide (4NQO) in drinking water.

Oral administration of 0.001% 4NQO in drinking water resulted in a high incidence of tongue carcinoma in rats. In other major organs, tumor induction was rarely observed. The most frequent site of tongue carcinoma was the dorsum. No metastases were found. Changes observed included carcinoma in situ and invasive carcinoma. Carcinoma in situ showed erosion, leukoplakia, and a gross papillary appearance. Histologically, most carcinomas in situ showed full-thickness alteration of epithelium. Some carcinomas in situ in papillary lesions showed slightly less than full-thickness alteration of epithelium, exhibiting downward, well-differentiated growth. Invasive carcinomas were either endophytic or exophytic. Histologic grading of invasive carcinoma varied from highly to poorly differentiated. The method described offers a new experimental animal model of tongue carcinoma.