Einfluß fünf verschiedener nicht-genotoxischer Karzinogene auf das Wachstum spontan entstandener präneoplastischer Herde in der Leber alter Wistar Ratten

Abstract

1. The aim of the investigation was the analysis of the impact of five different non-genotoxic carcinogens on the growth of spontaneously developed foci of altered hepatocytes (FAH) in the liver of old male Wistar rats (81-89 weeks). The chosen compounds represent well-known liver tumor promotors working through different mechanisms: phenobarbital, chloroform, Wyeth-14,643, ethinylestradiol and PCB 126. The rats were treated consecutively for 1, 4 and 13 weeks at doses which produce liver tumors after long term administration. Additionally, the effect of the substance application on cell proliferation in extrafocal rat liver tissue was analysed.

2. The number of FAH/cm², the average size of FAH (cell number/FAH-cross section), the labelling index (LI %) and the apoptotic index (AI %) were measured considering the different FAH-types for quantifying the growth of FAH. Cell proliferation in extrafocal hepatocytes was measured using the LZM-method (lobule zonal measurement) described by BAHNEMANN and MELLERT (1997).

3. FAH were identified and classified by histomorphological criteria (H&E) in combination with immunohistological detection of GST-P. The labelling index was determined in hepatocytes immunohistochemically by BrdU staining after implanting a 7-day osmotic minipump. The apoptotic index was established by counting the apoptotic bodies (H&E).

4. All animals investigated showed FAH. Clear cell foci (CCF) were the most frequent type of lesion (84-89 %) in the 3 control groups. Additionally, basophilic cell foci (BCF) and NAH were observed (4-8%). NAH are characterised by positive GST-P staining, but cannot be seen in H&E slides.

5. Only after application of ethinylestradiol a statistically significant promoting effect in form of an increase in the total number of FAH/cm² has been observed. On closer inspection of the different FAH-types a promoting effect was shown in an increase in the number of special FAH-types/cm² (phenobarbital and PCB 126: ECF (eosinophilic cell foci), Wyeth-14,643: BCF, ethinylestradiol: BCF and MCF (mixed cell foci)). Thus phenobarbital, Wyet-14,643 and PCB 126 promoted in old rats the same FAH-types described in initiations-promotions-studies.

6. With regard to the evaluated parameters the increase in cell proliferation exhibited the strongest clue to the promoting activity of the compounds. With the exception of PCB 126 all substances caused a peak in cell proliferation in CCF (Wyeth-14,643: 7.8-fold, ethinylestradiol: 4.6-fold, chloroform: 3-fold, phenobarbital 1.9-fold) after 1 week followed by a decrease. The CCF in animals treated with chloroform, Wyeth-14,643 and ethinylestradiol showed a sustained increase in cell proliferation for 13 weeks. BCF, that represent advanced preneoplastic lesions in the carcinogenic process with a high potential for malignant transformation, exhibited a higher LI than CCF after 1 week of administration in all substance groups (except of the Phenobarbital group).

7. The LI in FAH (with exception of NAH) exceeded the extrafocal LI at all time points of investigation. The course of the LI during the application showed parallels in tendency between CCF and the extrafocal liver tissue generally and for the other FAH-types in most cases. The focal hepatocytes seemed to react to the same extent as the extrafocal hepatocytes to the cell proliferation increasing effects of phenobarbital-, disproportionally strong to ethinylestradiol- and PCB 126-administration (only after 13 week administraion) and, to a somewhat lesser degree, to chloroform- and Wyeth-14,643-administration.

8. An inhibition of the apoptotic process in FAH as promoting mechanism has not been observed.

9. The five compounds caused different pattern of cell proliferation in extrafocal liver tissue. The association between zonal predominance of extrafocal cell proliferation and zonal localisation of FAH after treatment reflected the influence of extrafocal cell proliferation for the promotion of FAH by 4 of the 5 substances tested.