Melbourne Dental School - Theses
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ItemAscorbic acid deficiency and Harvey-ras proto-oncogene expression in experimental oral mucosal carcinogenesis( 1996)The aims of the present project were to examine the role of L-ascorbic acid (L-AA) deficiency and expression of the proto-oncogene, Harvey-ras (H-ras), in in vivo rodent oral mucosal carcinogenesis. The inbred, mutant Osteogenic Disorder Shionogi (ODS) rats used in the present study are unable to synthesise L-AA because they lack the liver microsomal enzyme, L-gulono-y-lactone oxidase (GLO), needed in L-AA biosynthesis. Foot length measurements and random analyses of microsomal GLO, by using rabbit anti-GLO rat-antiserum, were undertaken to confirm the breeding of ODS rats. The minimal L-AA requirement for survival of ODS rats under experimental conditions were determined. Two levels of L-AA deficiency were examined in ODS rats, while outbred Wistar rats acted as controls in the carcinogenesis experiment. 0.5% m/v 4 nitroquinoline-1-oxide (4NQO) in propane-1, 2-diol was used topically to induce oral mucosal squamous cell carcinoma (SCC) in ODS (n=72) and outbred Wistar (n=48) rats with vehicle and untreated controls (n=168). Harvested palatal tissues were analysed qualitatively, by using a modified dysplasia index, and histometrically. Total RNA extracted from rat palatal SCC (n=22) were analysed for mutations in codons 12, 13, 59 and 61 of H-ras by using reverse transcription (RT)-polymerase chain reaction (PCR) and direct DNA sequencing. H-ras mRNA expression was examined in 4NQO-induced rat palatal SCC and untreated palatal samples using semi-quantitative RT-PCR. Foot length measurements differed significantly (p«0.01) between ODS and outbred Wistar rats, confirming the genotype of the former. Likewise, rat GLO was detected in the positive outbred Wistar control, but not in the six random ODS samples. ODS rats were shown to require 10 mg L-AA/day for prolonged survival under experimental conditions. Plasma L-AA levels in ODS rats were adjusted to one-eighth of one-half that of the outbred animals (48±2 µM). The carcinogenicity of 4NQO was confirmed in ODS and outbred rats, with palatal SCC detected after 20 weeks and by the 40th week, respectively. Dysplasia scores for 4NQO-treated ODS rats were significantly greater than for 4NQO-treated outbred Wistar rats at all time periods over the 24 weeks (p<0.05), but were not significantly different between ODS rats with plasma L-AA levels of 7µM and 26 µM (p=0.94). The mean palatal epithelial thickness measurements increased significantly with time in all 4NQO-treated animals (p=0.04) and were significantly different between varying levels of epithelial dysplasia (p<0.05), but the relative proportions of individual epithelial compartments were maintained. No H-ras mutation was detected in the specified codons in any 4NQ-induced palatal SCC samples compared to untreated palatal mucosal samples. It was concluded that L-AA deficiency, in the model of chemical carcinogenesis used, did not influence the process of experimental carcinogenesis. The absence of H-ras mutations at specified sites and the observed H-ras mRNA levels in 4NQO-induced rat palatal SCC suggested that the H-ras proto-oncogene was unlikely to have contributed to malignant transformation in the present study.