Three isoforms of nitric oxide synthase (NOS) have been identified previously--endothelial NOS, neuronal NOS, and inducible NOS (iNOS). It has been reported previously that there may be a negative feedback loop existing between nitric oxide (NO) production and wild-type p53 tumor-suppressor gene, but the relationship has not previously been studied for oral experimental carcinogenesis. The purpose of the present study is to assess whether iNOS expression correlates with p53 expression at both protein and mRNA levels for 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch carcinomas.
Materials and methods: Thirty-five out-bred, young (6 week old), male, Syrian golden hamsters (Mesocricatus auratus) were randomly divided into one experimental group (15 animals), and two control groups (10 animals each). Bilaterally, the pouches of a group of 15 animals from the experimental group were painted with a 0.5% DMBA solution three times a week for 12 weeks whilst each animal from one of the control groups was similarly treated with only mineral oil. Another control group of 10 animals remained untreated throughout the experimental procedure. Specimens obtained from the hamster buccal-pouch mucosa were evaluated using immunohistochemical assessment of iNOS and p53 protein and in situ reverse transcription-polymerase chain reaction (IS RT-PCR), as well as reverse transcription-polymerase chain reaction (RT-PCR) for iNOS and p53 mRNA.
Results: Two of the 15 animals of the DMBA-treated group died during the experiment. Squamous-cell carcinomas with a 100% tumor incidence were apparent for all of the 15-week DMBA pouch-treated animals. Animals from the mineral oil-treated and untreated pouch groups revealed no obvious changes. Inducible NOS mRNA was identified as a band corresponding to a 499-bp PCR product and was observed for all 13 of the hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks. The p53 mRNA was found as a band corresponding to a 370-bp polymerase chain reaction (PCR) product and was noted for nine (9/13, 69%) of the 15-week DMBA-treated pouches. No such bands (iNOS and p53) were noted for the untreated animals, the mineral oil-treated tissues and the negative-control samples. Using IS RT-PCR, the proportional (percentage) expression of iNOS (13/13, 100%) and p53 (8/13, 62%) mRNA observed for the hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks was noted to be consistent with the findings using RT-PCR. Furthermore, the proportional expression of iNOS (13/13, 100%) and p53 (8/13, 62%) proteins for the 15-week DMBA-treated hamster buccal-pouch tissue specimens was noted to be consistent with the findings using RT-PCR and IS RT-PCR. A significant association between iNOS and p53 expression (at both protein and mRNA levels) was noted (Fisher's exact probability test, P < 0.05). Neither iNOS nor p53 activity (at both protein and mRNA levels) was found for any of the untreated and mineral oil-treated pouches.
Conclusions: Enhanced expression of iNOS and p53 at both protein and mRNA levels in DMBA-induced hamster buccal-pouch carcinomas compared with the untreated and mineral oil-treated counterparts, has been demonstrated in the current study. Furthermore, we report what is, to the best of our knowledge, the first identification of a significant association between iNOS and p53 expression (at both protein and mRNA levels) in this experimental model system for oral carcinogenesis, although their precise interactions remain to be clarified.