The influence of estrogen on liver regeneration . An experimental study in rats

Purpose: To recognize the regenerative capacity influenced by the administrating of estradiol. Methods: 42 female Wistar rats were used, divided into two groups, the control and the experiment group. A resection of approximately 70% of the liver was made in the liver of these animals. The control group received an intramuscular injection of one ml of peanut oil. The experiment group were given estradiol hexahydrobenzoate (50μg) diluted in one ml of peanut oil. Calibrations were done after 36 hours and 7 days, using three methods: the formula of Kwon et al.21, to recognize gain in volume, counting of the mitosis figures in five fields and the percentage of positive PCNA nuclei. Results: Gain in volume (mass) was similar in both groups after 36 hours (p=0.1873) and higher in the experiment groups after seven days (p=0.0447). Microscopy showed a similar number of mitosis figures after 36 hours (p=0.3528) and a tendency to be higher in the experiment group after 7 days (p=0.0883). The average of positive PCNA nuclei was higher in the experiment group both after 36 hours (p=0.0009) and 7 days (p=0.0000). Conclusion: The estradiol hexahydrobenzoate improved liver regeneration in rats submitted to a 70% hepatectomy.


Introduction
Liver regeneration capacity has been known for centuries.It is a complex process involving cytokines and hormones.
Although commonly used, the meaning of the term regeneration is only that of restoring the volume of the viscera and not strictly its initial appearance.In truth, what happens is hypergenesis with compensatory hypertrophy of the remaining segments until the primitive liver mass has been reestablished 2,3,4 .
The process is an organic protection mechanism against loss of functioning liver tissue be it by chemical, viral or traumatic aggression or surgical resection 5,6,7 .There is highly ordered and organized tissue growth until the liver regains its original weight, with a small variation of 5-10%.However, there is a period of transitory liver insufficiency during the recomposition of the liver mass, which has led to the growing search for factors capable of influencing hepatocellular proliferation and the regenerative process 8,9 .
There seems to be a clear distinction between the cells that originate the new hepatocytes and after liver resections and those which originate following cellular necroses with acute liver insufficiency.After hepatectomies, regeneration is done by the replication of the normally quiescent hepatocytes, without activation of the progenitor cells, while regeneration after losses induced by toxins occurs through the replication and differentiation of intra-hepatic progenitor cells (oval cells) 10 .
The anxiety of accelerating agents in the regeneration process is justified by the importance of a short period of hepatic insufficiency following a hepatectomy, which would lead to much wider resections.
There is evidence that suggests estradiol plays an important role in liver regeneration.In male rate, the plasma concentration of estradiol is increased in the first six hours following a hepatectomy [11][12][13] .It has been suggested that this may play an important role in facilitating initial liver regeneration 14 .Fisher et al. 15 report that the administration of 17-beta-estradiol induced the rapid beginning of translocation of estrogenic cytoplasm receptors to the nuclei of the parenchymatous cells 15 .The DNA synthesis is increased following the nuclear internalization of the receptors 15 .Francavilla et al. 16 and Klatskin et al. 17 found partial blockage of regeneration after the administration of tamoxyphen [16][17] .Mizushima et al. 18 observed that the administration of estrogens in rats following hemorrhagic trauma improved the functioning of the liver.It is interesting to note that the administration of estradiol reduces the seric level of interleukin-6 and the non-offering of estradiol led to the raising of the levels of interleukin-6 18 .It seems logical to think that there is a feedback between IL-6 and estradiol.
Chiu et al. 19 demonstrated that patients with liver function deficiency, following hepatectomies, saw an improvement in their function when treated with estradiol.
Although it is acknowledged that estrogens do indeed stimulate liver regeneration, a search for this information in the literature turns up very few results and thus the interest to know its real importance.
This paper sought to establish a more precise relationship of the effect of estrogen on liver regeneration by seeking to recognize the regenerative capacity of the liver of animals treated with estradiol and compare them to the animals in the control group.

Methods
The project which originated this study was submitted for analysis by the Ethics in Health Sciences Research Committee at the Federal University of Paraná and approved and allocated the number CEP/SD:NA.010.001.08.1 house in a light-dark cycle with the natural humidity and room temperature of the environment, with free access to water and normal food.
The sample was divided into two groups, control (C) and experiment (E).There were 18 rats in the control group and 24 in the experiment group.Under an anesthetic of 0.1ml/100g of weight and a mixture of 1ml of ketamine (50mg) and 1ml of xylazine (20 mg), the animals were submitted to trichotomy of the ventral abdominal wall and antisepsis with poly vinyl pyrrolidone-iodine.A median laparotomy was the performed and a partial hepatectomy, with resection of the median and left lateral lobes.This hepatectomy is equivalent to a resection of approximately 67% of the viscera volume 20 .The resected segments were weighed and noted.Following the laparorrhaphy, the rats in the control group were given an intramuscular injection of one ml of peanut oil, while the experiment group were given estradiol hexahydrobenzoate (50µg) diluted in a ml of peanut oil, every 24 hours until calibration.These were carried out after 36 hours and seven days when ten animals from each group were submitted to euthanasia.In this way the subgroups C 1 , C 2 , E 1 e E 2 were obtained.The rats were then submitted to another laparotomy with total resection of the liver, which was weighed and the measure noted on the chart.
The resected parts were set in covered formalin at 10% and forwarded for histopathological study, with the histological cuts colored by hematoxylin and eosin for the counting of mitosis figures in five fields and by immunohistochemicals through the use of PCNA (cellular proliferation nuclear antigen) for the count of positive and negative nuclei and DNA replication in five fields.
Regeneration was evaluated using three methods: the formula of Kwon et al. 21, by the count of mitosis figures in five fields and by the count of positive PCNA nuclei in five fields.The formula of KWON et al. 21provides the rate of regeneration based on weight.

% = D/E .100
Where E = R/0.7 and D = weight of the liver per 100g of the weight of the animal when sacrificed, with "E" being the estimated weight per 100g of animal weight before the partial hepatectomy, calculated from the weight of the resected liver (R).

Results
One death was registered in the control group after seven days.
Gain of mass was similar for both groups after 36 hours (p=0.1873) and higher in the experiment group after 7 days (p=0.0447)(Table 1  The histological cuts revealed the number of mitosis figures in the 36-hour evaluation with average values of 2.78 ± 0.83 for the control group and 2.64 ± 0.81 for the experiment group (p=0.3528).In the evaluation after seven days, the average for the control group was 5.89 ± 0.78 and for the experiment group it was 5.18 ± 1.33 (p=0.0883)(Figure 1).

FIGURE 1 -Average number of mitosis figures at both times
The average of positive PCNA nuclei was higher in the experiment group both after 36 hours (p=0.0009) and 7 days (p=0.0000).The values of the averages obtained were 10.44 ± 2.96 for the control group and 14.00 ± 1.26 for the experiment group after 36 hours and 6.00 ± 0.87 for the control group and 10.09 ± 2.51 for the experiment group after 7 days (Figure 2).

Discussion
It has been suggested that estrogen may play an important role in facilitating the beginning of liver regeneration 14 .Estrogens have recognized hepatotropic effects 15 like a role in liver carcinogenesis 22 .
While a little exposure to 17 -ethinilestradiol can stimulate liver growth, in rats 23,24 prolonged exposure inhibits the proliferation of hepatocytes 25 .17 -ethinil-estradiol reduces the number of cells in the S phase and in the mitosis, suggesting previous blocking of the DNA synthesis 26 .
With the assumption that exposure to estrogen for short periods might aid liver regeneration, Chiu et al. 19 administered estradiol to patients with liver function deficiency after hepatectomies and found that In this study, the administration of estradiol hexahydrobenzoate determined a higher number of positive PCNA hepatocytes at both times studied, although this number was higher after 36 hours.The number of mitosis figures proved to be higher after seven days in both groups, with no significant difference between one group and the other.The regenerated liver mass was similar in both groups after 36 hours (p=0.1873) and higher in the group that was given estradiol hexahydrobenzoate after 7 days (p=0.0447),corroborating the same positive relationship reported by Ramalho et al. 5 , one of the positive factors described in the literature.It is possible that these results attribute to estradiol a beneficial effect for liver regeneration that is more significant in the early stages, an initial facilitation already suggested by Liddle et al. 11,12 .

Conclusion
Estradiol hexahydrobenzoate improves the liver regeneration of rats submitted to a 70% hepatectomy.their function improved 10 .Kopplow et al. 27 demonstrated that dehydroepiandrosterone is mitogenic, but reduces the regenerative capacity of the liver 27 .The administration of tamoxiphen, however, led to partial blockage of the liver regeneration 12,13,17 .The liver cells have estrogenic receptors.Thus the administration of tamoxiphen, by competing with the estrogens for the receptors, would account for the blockage of the regeneration.
Hepatectomies are accompanied by the elevation of the seric levels of estradiol 11 .The administration of estradiol to rats with abdominal trauma reduces the seric level of interleukin-6 and the non-offering of estradiol led to the elevation of the levels of interleukin-6 18 .Interleukin-6 is a cytokine produced by cells with immunological activity such as macrophages, T cells and the Küpffer cells of the liver.This cytokine promotes liver regeneration during chronic lesions and after hepatectomies.In rats, the level of interleukin-6 increases in the first hours following hepatectomy to 2/3 and reaches its peak after 24 hours.It was reported that the reduction of interleukin-6 lowers liver regeneration after partial hepatectomies 28 .

FIGURE 2 -
FIGURE 2 -Demonstration of the average of positive PCNA nuclei in the two groups and on both two occasions of evaluation ).

TABLE 1 -
Percentage of liver mass gain at the two times studied in the control and experiment group