Stereological Analysis of gamma-Glutamyl Transpeptidase Positive Foci in Diethylnitrosamine-Induced Hepatocarcinogenesis of Sprague-Dawley Rats

Yoo, Yong Keol; Kim, Hong Joo; Lee, Soong Hwan; Gham, Chang Woo; Kim, Hee Soo; Kim, Jong Cheol; Nam, Seung Woo; Kim, Sung Woo; Kim, Byeong Hoon; Yang, Sung Kyu; Park, Dong Il; Lee, Dong Hoo

Abstract

BACKGROUND/AIMS
Hepatocarcinogenesis of microscopically altered foci could be shown to be progressed into a trabecular pattern of hepatocellular carcinoma. And it is reported that down-regulation of TGF beta II receptor and up-regulation of TGF alpha and c-myc reveal the progression of diethylnitrosamine-induced foci into liver cell cancer. Up-regulation of TGF beta II receptor, however, causes apoptosis of foci. To determine characteristic morphology and growth kinetics of putatively precancerous y glutamyl transpeptidase (GGT) positive foci and hyperplastic nodules, a stereological quantification was attempted in the Peraino's neonatal rat model initiated by diethylnitrosamine and promoted by phenobarbital.
MATERIALS/METHODS
Fifteen Sprague-Dawley rats were I.p. injected with 0.15 pmole/g of body weight of diethylnitrosamine mixed in corn oil at one day after birth. From weaning at 4 weeks of life, the rats were continuously fed 0.035% phenobarbital in drinking water and sacrified 5 rats at each time point of 8 weeks, 16 weeks, and 32 weeks. Teklad standard diet was fed after weaning. The livers obtained were fixed in freshly prepared, cold ethanol-acetic acid (99:1 vo1%). For the GGT histochemical staining, Rutenberg's method was modified, and counterstained with H & E or toluidine blue. For the stereological analysis GGT positive foci and nodules were traced in 200 consecutive tissue sections and quantified the 3 dimensional volumes by computer assisted planimetry. Either spheroidal or non-spheroidal morphology was determined by parabola 2nd degree equation ' y=ax+bx+c (sphere a=-P,).
RESULTS
Thirty nine (55.71%) out of 70 representative lesions were nonspheroidal. Especially at 8 weeks, the 28 out of 40 GGT positive foci were irregular, nonspheroidal shape. Later times, however, GGT positive foci and reprogrammed nodular lesions were become spheroidal. Lilliefors probabilities test for spheroidal frequency was statistically significant (p<0.05).
CONCLUSION
Stereologically non-spheroidal characteristics of the early GGT positive foci limit growth kinetic estimation by 3 dimensional volume quantitation but permit in later times in spheroidal, GGT positive foci and reprogrammed nodules showing fade-out of GGT activity. In other words, GGT positive foci may be clonally selected for growing into hyperplastic nodules and hepatocellular carcinoma or regressed by apoptosis.