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Studies with Diethylstilbestrol (DES) in humans and rodents have resulted in a spectrum of toxic and carcinogenic effects. Previous findings on gene expression profiles following DES treatment showed that p53, p21 and bax was transcriptionally regulated in this model (Salleh et al.,2003). In the present studies, we used Reverse Transcriptase in situ Polymerase Chain Reaction (RT in situ PCR) and immunohistochemistry techniques for localisation and expression of p53, p21 and bax at cellular levels. “Metabonomic approach” based on 1H-NMR spectroscopy also has been applied to investigate metabolic profiles of the biochemical effects of clastogenic carcinogen (DES). Animals were housed individually and treated with 500μmole/kg b.w of DES, (ip) once daily up to four days. Urine was collected at different time-point (pre-dose, 0-8, 8-24, 24-32, 32-48, 48-56, 56-72, 72-80 and 80-96 h) and control animals were treated with the trioctanoin vehicle only. Our results have shown, the expression of p53, p21 and bax mRNA were greater in wild-type compared to p53+/− knockout mice. In addition, p53, p21 and bax mRNA were significantly high in DES-treated compared to control-vehicle animals. Collectively, similar patterns of expression also were seen in p53 and p21 proteins and scored according to the percentage of positive nuclear staining. Chemometric analysis of 1H-NMR data have shown that there are modification changes of metabolite between DES-treated and control-vehicle animals. Biochemical changes include increased urinary levels of creatine, lactate, taurine, hippurate and reduced levels of tricarboxylic acid cycle (TCA) especially 2-oxoglutrate and trimethylamine-N-oxide levels in wild-type DES-treated compared to p53+/− mice. The elevated taurine and creatine levels in the urine of treated animals were due hepatocyte necrosis as shown in histological examination. Therefore, the combination of p53 and p21 were concluded to be a good prognostic marker for development of carcinogenesis and the 1H-NMR spectroscopy, is a new view to generate diagnostic tools for the early detection of toxicity in this model.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]