Tel.+49 30 838 62550 Fax.+49 30 838 46029
With regard to tumorigenic activity, the change in the growth kinetics of hepatocytes following long-term high-dose administration of mitogenic steroids to rats has not as yet been investigated in situ on the basis of the simultaneous determination of proliferation and apoptosis. Therefore the aim of the present study is for the first time to investigate simultaneously proliferation and apoptosis in the liver parenchyma on the basis of label lingindices and with respect to the formation of liver cell foci.The effect of the progestin gestodene on he liver is, as is usual in tumorigenicity studies, investigated following daily intragastric application, in this case to 216 female HAN Wistar rats. The animals are 10 weeks old at the start of the study, as recommended by GOLDSWORTHY et al. (1991). In addition to the control group which is treated with 0 mg/kg/d gestodene (n=72, group 1), four groups are formed for treatment. The dosages of 0.2 mg/kg/d (n=36, group 2), 2 mg/kg/d (n=36, group 3), 20 mg/kg/d (n=36, group 4) and 100 mg/kg/d (n=36, group 5) gestodene are selected. This is expected to lead to minimal to maximal biological effects which can be interpreted in connection with data from the literature (carcinogen i city study: max. 0. 1 25 mg/kg/d gestodene over 104 weeks, tolerance study: max. 1 mg/kg/d gestodene over 26 weeks, short-term study: max. 67mg/kg/d gestodene over seven days). Am groups are allocated to one of three collectives, as recommended in the literature, which are defined here according to timepoint of sacrifice after 2, 20 and 39 weeks. The proliferation indices are detected using the currently common BrdU-method, th% apoptosis indices using the new TUNEL-technique separately for the unchanged parenchyma and the liver cell foci which are detected. In previous studies the BrdU-method was optimised on the basis of staining results and the TUNEL-technique was used for the first time by means of a TUNEL-kit not yet tested on rat hepatocytes and optimised for the present study. For the systemic, continuous BrdU release a minipump is implanted subcutaneously in the animals of all collectives 15 days before necropsy and not seven days as in the prestudy, in order to increase, sensitivity. As described in the literature, liver weights and DNA contents in the liver are recorded separately. In addition body weights as well as food and waterIQ Aconsumption are also determined, as is usual. The common assessment of clinical findings, of histological investigation of livers after HE-staining and determination of serum transaminase (ALAT, AP) serve the detection of toxic organ damage. On the fourteenth day of treatment with gestodene, besides the collective 1 of group 5 which is to be killed according to plan, collectives 11 and Ill of group 5 are also killed earlier than planned due to unexpected severe general intolerance reactions.The proliferation index in the unchanged liver parenchyma is dose-dependently increased after two weeks on average from 5.5 % (group 1) o 9.0 %, 18.6 % and 30.6 % (groups 2, 3, 4), confirming the mitogenic potential described in the literature; the cumulative proliferation detection does not support the burst-like progress to be derived from information given in the literature. After 20 weeks all values are unexpectedly low at the control level. After 39 weeks, moreover, the mean proliferation indices are dose-dependently decreased from 3.8 % (group 1) to 2.5 %, 1.7 % and 1.5 % (groups 2, 3 and 4). These results contrast with the increased proliferation after four- to forty-week administration of ethinyl estradiol. The apoptosis indices in the unchanged parenchyma are not influenced by gestodene. On the other hand, the literature describes apoptosis inhibition at least after short-term administration of various mitogens. The time-dependently decreasing apoptosis and proliferation indices indicate time-dependently decreasing cell turnover as opposed to increased cell turnover following administration of ethinyl estradiol. The close- and time-dependent increase in liver weights and DNA-contents arises from initial proliferation increase as well as from reduced proliferation with decreased proliferation rate resulting from raised cell level in the further course of the study (timedependently failing proliferation indices). These data confirm information in the literature. In comparison to the tumorigenicity study with gestodene liver cell foci already occur after 20 and 39 weeks, whereby they first appear after the very high dose of 20 mg/kg/d gestodene in contrast to the considerably lower doses stated in the literature. The present study is the first to provide information on the foci count as well as the indices of proliferation and apoptosis in the liver cell foci investigated. After 20 weeks sporadic liver cell foci with extremely high proliferation indices of 79 % to 92 % are visible without detectable apoptosis. The liver cell foci count is severely increased after 39 weeks with individually differing severity. In these liver cell foci the proliferation indices at 22 % to 79 % are clearly lower than after 20 weeks. Due to low apoptosis indices (0. 17 \% to 0.97 %) the cell turnover of the livercell foci is noticeably shifted towards proliferation. Severely increased proliferation confirms the severe clonal growth potential of liver cell foci described in the literature. Following histopathological liver examination and determination of serum enzymes it is possible to exclude regenerative processes as the reason for proliferation. This confirms expectations from information in the literature. In the light of knowledge from he literature the effects observed in the liver in this study are interpreted as being the result of a hepatomitogenic effect of gestodene which is known to be non-toxic.From the results of the present study in connection with data from the literature (tumorigenicity study) it can be assumed that gestodene has an influence on the liver cell turnover depending on the dose and, above all, the duration of application. Initially a severe adaptive liver growth which is known to be characteristic of mitogenic steroids is induced. After long-term application of up to 20 mg/kg/d gestodene the proliferation rate in the unchanged liver parenchyma falls (with supposed increase in cell count level) to below control level. It can be concluded from the literature that the liver cell foci discovered represent an increase of probably spontaneously occurring, initiated hepatocytes. After 20 and 39 weeks despite decreased mitosis rates in the unchanged liver parenchyma they show severe clonal growth which has up to now only been described after steroid-induced permanently increased parenchyma proliferation. According to the literature a time-dependent increase in the number of liver cell foci indicates an increased phenotypic expression, which is described as a possible early stage in the development of tumours. The nonoccurrence of tumours can be explained in the present study by the unexpected retrograde proliferation rate in the liver cell foci as well as by the short duration of application of 39 weeks in comparison to the tumorigenicity study (104 weeks). On the other hand, in this case, a low apoptosis rate in the liver cell foci would rather suggest tumorigenesis. Tumour-promoting activity assumed for gestodene in the literature can not be confirmed in the present study. Since the growth of liver cell foci was more intensive after 39-week application of gestodene, in the present study gestodene is regarded only as a promoter of liver cell foci in female rat liver.