WILD-TYPE P53 EXPRESSION IN LIVER-TISSUE AND IN ENZYME-ALTERED FOCI -AN IN-VIVO INVESTIGATION ON DIETHYLNITROSAMINE-TREATED RATS

Previous reports have documented an attenuated p53 response to DNA dam age in hepatocytes isolated from enzyme-altered foci (EAF), Here, we h ave studied this p53 response in vivo in rats with EAF, These animals received repeated doses of diethylnitrosamine (DEN) for 6 weeks and a challenging dose 24 h before death. Liver sections were then analysed using an immunohistological procedure for p53, or a double-staining pr ocedure for p53 and glutathione-S-transferase pi (GST-P), In control r ats or rats,vith EAF not given the challenging dose of DEN, there was no p53 staining. In control rats, only given the challenging dose of D EN, there was a centrilobular p53 nuclear staining that co-localized w ith TUNEL staining. Tn an experiment involving four rats with EAF 389 +/- 39 hepatocytes/mm(2) of non-LAP tissue stained positively for p53, while the corresponding value for EAF tissue was 27.6 +/- 7.5, Thus, p53-positive cells were 14.6-fold more frequent in non-LAP than in EAF tissue. In many EAF no p53-positive cells were seen at all and 83% of the EAF demonstrated <20% of the number of p53-positive cells seen in non-LAP tissue. Very few EAF had as high a proportion of p53-positive cells as did the average non-EAF tissue, EAF >0.06 mm(2) had signific antly fewer p53-positive cells than smaller EAF, The ratio of p53 expr ession in non-LAP tissue and large LAP was 32.6, In a control experime nt, four LAP-hearing rats were used as donors to prepare primary cultu res of hepatocytes. After 24 h of exposure to DEN, many of the culture d cells became p53-positive. Among GST-P-negative hepatocytes, 12.8% w ere p53-positive, whereas only 0.25% of the GST-P-positive hepatocytes were p53-positive. Literature data suggest that the altered xenobioti c metabolism in EAP may give rise to a 3-4-fold difference in DNA dama ge between non-LAP and LAP tissues. It is concluded that GST-P-positiv e EAF hepatocytes have an attenuated p53 response to DNA damage, This attenuated response may facilitate clonal expansion of LAP under stres s induced by DNA-damaging chemicals.