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Quantitative Stereological Analysis of the Effects of Age and Sex on Multistage Hepatocarcinogenesis in the Rat by Use of Four Cytochemical Markers¹

McArdle Laboratory for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin 53706 [H. A. C., G. L. S., S. H., H. C. P.]; Department of Pathology, Shanghai Medical University, Shanghai 200032, People's Republic of China [Y. X.]; National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [R. M.]; and Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki 036, Japan [K. S.]

Altered hepatic foci (AHF) were analyzed by quantitative stereology on frozen serial sections stained sequentially for -glutamyltranspeptidase (GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6Pase), and the placental isoenzyme of glutathione S-transferase (GST). Livers for these analyses were obtained from both male and female rats of different ages which had been subjected to initiation with a nonnecrogenic dose of diethylnitrosamine following a 70% partial hepatectomy with subsequent phenobarbital (PB) feeding. Different combinations of these four marker alterations (from single marker to four-marker combinations) were used to analyze the data, and the results were compared for their ability to detect AHF. In rats on the above protocol, GST was the single most effective marker, exhibiting a high sensitivity for scoring both number and volume of foci. There was a high degree of overlap with GGT. The combination of the four different markers, GST/GGT/ATPase/G6Pase, scored 80% more foci in number and 60% more in volume than the routinely used GGT/ATPase/G6Pase method.

When all four markers were used to score AHF, PB promotion was equally effective in both sexes at weaning and at 6 months of age, but at 1 year of age males showed a dramatic reduction in the effectiveness of PB as a promoting agent, both for number and volume percentage of liver occupied by AHF. On the other hand, initiation was more effective in the male at weaning and at 6 months of age, although by the 12-month point no distinction between the sexes could be made. When only GGT was used as a marker, promotion by PB appeared to be markedly less effective in males than in females at all ages. In the absence of PB administration, both the number and volume fraction of AHF in the livers of both males and females increased with age. Likewise, both the number of AHF per liver and their volume fractions increased with age in both sexes when uninitiated animals were fed PB, although only after a 6-month lag in females. These experiments demonstrate that the stages of initiation and promotion in hepatocarcinogenesis in the rat as monitored by the number and volume percentage occupied of AHF are altered by both the age and the sex of the animal. The combination of GGT and GST identified all AHF scored by the GST/GGT/ATPase/G6Pase set of markers and thus may be the most efficient combination of markers of AHF resulting from promotion by PB.

¹ Studies described in this paper were supported in part by grants from the National Cancer Institute (CA-07175, CA-22484, and CA-45700) as well as a contract from the National Toxicology Program (ES-82-12) and a training grant from the National Cancer Institute (CA-09451).

² Present address: Department of Food and Nutrition, Iowa State University, Ames, IA 50011.

³ To whom requests for reprints should be addressed, at the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706.

Received 6/15/89. Revised 9/14/89. Accepted 10/24/89.

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