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| The Turkish Journal of Gastroenterology | |
| 2001, Volume 12, No 1, Page(s) 6-12 | |
| [ Summary ] [ Similar Articles ] [ Mail to Editor ] | |
| Effect of 5-Fluorouracil on liver regeneration and metabolism after partial hepatectomy in rat | |
| Köksal Hakan M.1, Erdem Levent2, Yıldrım Sadık1, İpek Turgut3, Pakiş Işıl4 | |
| Şişli Etfal Teaching and Research Hospital, Departments of General Surgery1and Gastroenterology2, İstanbul University Cerrahpaşa Medical Faculty, Departments of General Surgery3 and Pathology4, İstanbul. | |
| Keywords: Hepatic regeneration, hepatectomy, 5-fluorouracil, Hepatik rejenerasyon, hepatektomi, 5-fluorouracil. | |
| Summary | |
Background/aims: In this study, the effects of 5-fluorouracil (5-FU) on liver regeneration was studied after two thirds hepatectomy in rats, in vivo. Extensive hepatic resection is performed with increasing frequency for malignant tumors of the liver and administration of chemotherapy immediately after removal of the major tumor is favorable. Methods: Wistar male Albino rats, weighing 150-280 g, underwent hepatectomy under standard conditions. In Group A, 70% hepatectomy alone was performed. In Group B, 5-fluorouracil at a dose of 20 mg/kg was administered intraperitoneally and in Group C, a 30 mg/kg dose of 5-fluorouracil was administered intraperitoneally immediately after 70% hepatectomy. The rats were sacrifized on the 1st, 3rd, 5th and 7th days following hepatectomy. Argyrophilic nucleolar organiser region score, mitotic figure, Kuppfer cell counts and liver function tests were carried out. Results: Administration of 5-fluorouracil caused substantial suppression and delay of liver cell division in a dose dependent manner, especially on the 1st and 3rd day after hepatectomy. The effect of 5-fluorouracil reversed within 24 hours of drug withdrawal and arrested cells at the S phase completed the sequences. Conclusion: Treatment with 5-FU following hepatectomy caused not only suppression but delay of liver cell division.The results indicate that adjuvant chemotherapy with 5-FU after partial hepatectomy should be prescribed with great care or should not be carried out until hepatic regeneration is almost completed. |
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| Introduction | |
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The ability of the liver to regenerate remains a fascinating response to hepatic injury. Ever since the Greek myth of Prometheus, efforts have been made to unravel the mechanisms involved in liver regeneration. The cellular phenomenon represents an orchestrated response to external stimuli followed by sequential changes in gene expression, cytokine production, and morphologic structure and to many endogenous factors affecting the regenerative capabililty of the liver (1,2). The most popular experimental model to induce regeneration is based on the surgical removal of two-thirds of the liver (1-3). The remnant lobes respond to the loss of mass and function with expression of immediate- and delay-early genes which prime the cells for eventual progression through the cell cycle (4-6). The molecular events which trigger liver regeneration are now beginning to unfold. However, the control of liver regeneration and the events involved in regulating the three-dimensional growth of the organ remain poorly defined.
Partial hepatectomy triggers a variety of biological phenomena, which culminate in regeneration of the liver mass. Although hepatocyte proliferation and tissue remodelling are a major feature of the regenerating liver after partial hepatectomy, many other cell types (Kupffer cell, stellate cell, epithelial cells), cytokines (interleukin 6,8 etc.) are and factors (hepatocyte growth factor, vascular endothelial growth factor etc.) involved in an autocrine and paracrine way w(4,7-9). The hepatic regenerative process is triggerred with the resection of primary liver tumors, metastatic deposits and with any injury. Mean argyrophilic nucleolar organiser region (Ag-NOR) values help to evaluate the degree of malignant degeneration and some neoplastic and regenerative conditions like hepatic regenerative cell kinetics. Different tissues or cell lines have different Ag-NOR amounts and morphology (10-14). Valuable information about proliferative activity of cells can already be gained by counting the Ag-NOR number per nucleus. The number of interphase Ag-NORs is strictly related to rRNA transcriptional activity and, in continuously proliferating cells, to the rapidity of cell proliferation. Moreover the use of digital image analysis provides information about size and distribution of Ag-NORs. Chemotherapy against the cancer is normally commenced after resection of the tumor during the convalesence period, while the liver is regenerating (5). 5-Fluorouracil (5-FU) is the antimetabolite drug of choice and is used in many regimens. Additionally, due to the fact that 5-FU based chemotherapy and radiotherapy have become standard treatments at a variety of tumour stages in the last 10 years, the outstanding predictive power of AgNOR analysis with regard to chemotherapy efficiency is emphasised (15). Prior studies have suggested that changes in liver function tests may vary with the postoperative time interval and may be related to the extent of hepatic resection (16). Surgical resection remains the only potentially curative treatment for hepatic malignancies and is the gold standard against which all other therapies are compared. The human liver regenerative period has been calculated by various methods and is estimated to last from six to twelve months (17-19). It would be ideal if chemotherapy was administered immediately after the removal of the hepatic neoplasm to prevent recurrence. This experimental study was designed to investigate the effect of phase specific antimetabolite 5-FU on hepatic regeneration after two-third hepatectomy, mimicking the same phenomenon in humans following resection due to metastases and related hepatectomies. Table 1: Results of mitotic index
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| Materials And Methods | |
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In this study, Wistar-albino rats weighing 150-280 g were used and they were kept in standardized conditions at room temperature in a cage. The rats were divided into three main groups (Groups A, B and C) comprising 20 rats each. In group A, two-third hepatectomy was performed and the rats were given normal saline intraperitoneally. In group B rats, 20mg/kg of 5-FU was given intraperitoneally and in Group C, 30mg/kg of 5-FU was given intraperitoneally immediately after 70% hepatectomy and before the abdomen was closed. Each main group was divided into four sub-groups of five rats according to the day they were killed. All operations and sacrification of the rats was performed on the 1st, 3rd, 5th, and 7th day postoperatively (Groups A1-A3-A5-A7, B1-B3-B5-B7, and C1-C3-C5-C7) and was carried out in the morning between 09.00-12.00 a.m. to minimize the hormonal effects on regeneration. Operation and killing was performed under light ether anesthesia. In the preoperative period all rats were weighed to calculate the dose of the 5-FU.
Hepatectomy was performed according to Higgins and Anderson’s definition: median lobe and left lobe of the liver are freed respectively and their pedicles tied with silk sutures; mean operation time was five minutes (10). All rats were killed on predetermined days and a blood sample taken from the right ventriculi for analysis of serum biomarkers of hepatocelluler necrosis: levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST), alkaline phosphatase (ALP), markers of hepatocelluler regeneration gamma-glutamyl transferase (GGT) and bilirubin and to remove tissue from the regenerating liver. Argyrophilic nucleolar organizer region score (the mean number of AgNORs / 100 cell nuclei), mitotic index (MI), cell counts with hyperchromatic nucleus and double nucleolus and Kupffer cell (KC) counts were calculated. After the resection of regenerating fresh liver tissue to obtain Ag-NOR score, imprinting method was used. Statistical comparisons between groups and results from each day of killing were made using the chi-squared analysis and Student’s t-test as appropriate, differences between groups with the same days of death were performed using multivariance analysis, as appropriate, with statistical significance defined as P<=0.05. |
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| Results | |
No rats died in Group A (control group) throughout the experiment (mortality rate 0%). Four rats died in Group B: two on the first, one on the second and one on the fifth postoperative day (mortality rate 20%), while 11 animals of 20 in Group C died: six on the first, three on the second, one on the fourth and one on the fifth postoperative day (mortality rate 55%).
As shown in Table 1, the peak increases of mitotic index (MI) in each group were seen on the third postoperative day. Fifth and the seventh day MI values were higher than in the groups administered 5FU than the controls. On the seventh day, mitotic figure was still observed in Group C and results were higher than Group B. There were significant differences between groups on the third postoperative day (p = 0.05) and the seventh postoperative day (p = 0.0472). Regarding Kupffer cell count results (Table 2), there seemed to be substantial differences: for the first postoperative day between Group A and Group C (p=0.001), Group B and C (p=0.013) and Group A, B and C (p=0.0004). The KC counts of Group C were lower than the others and the highest counts were obtained from the control Group A. For Ag-NOR score, maximum levels were seen in Group A. On the first postoperative day, peak levels later steadily declined to levels regarded as normal seven days after partial hepatectomy, and these levels obtained on day seven in Group A were observed on the first postoperative day in Groups B and C. Ag-NOR scores of Groups B and C were different in pattern. In Group B, peak scores were obtained on the fifth postoperative day, whereas in Group C they were obtained on the third postoperative day. (Table 3) For the same postoperative day results, substantial differences were seen. The Ag-NOR scores were significantly higher in Group C on day three and seven than the other two groups (p=0.0264 and p=0.0167 respectively). Comparisons were as follows: Group B on day 3 (p=0.027) and on day 7 (p=0.015), Group A on day five (p=0.030) and on day seven (0.050). In Group B Ag-NOR levels were the lowest on they three compared to controls (p=0.046). Changes in ALT, AST, GGT, ALP and bilirubin levels were not significant in Group A except when day three and day five AST results (p=0,024), and day three and five ALP results (p=0.024) were compared, whereas in study Group B and especially in Group C, 5-FU caused several significant results for ALT, AST, ALP and GGT values. Negligible results were obtained for serum total and direct bilirubin levels. However, the apparent changes and significant results in AST, ALT, GGT and ALP levels did not correlate with the results obtained for conventional histochemical values. In summary, treatment with 5-FU caused not only suppression, but also delay of remnant liver regeneration. Table 4 and 5 show the mean standard devration (SD) and standard error SE values of the parameters that were used in this study. |
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| Discussion | |
It is common surgical practice today for primary and metastatic hepatic neoplasms with the primers to be resected, due to improved life-expectancy. Synchronised liver resections can be safely performed without endangering the gastro-intestinal anastomosis (17, There are several methods for assessment of proliferative activity of regeneration of the liver, the first being determination of MI, which has played an important role as a part of various grading systems to date. Other methods include DNA cytometry, flow cytometry, determination of bromodeoxyuridine labeling index (S phase) and immunohistochemical analysis of proliferation-associated antigens (e.g. Ki-67, PCNA expression (G (1) phase)). For several years, AgNOR analysis (Interphase-G(2)) has been used as a new technique, giving reproducible, exact results of proliferative activity of hepatic regeneration (2,27,28). Heat-labile growth inhibitory factor is released from Kupffer cells and would appear to act on hepatocytes in a paracrine manner, so the number of Kupffer cell or their function must be taken into consideration (29). 5-Fluorouracil is an S-phase specific drug. When 5-FU is added in a medium, proliferating cells then acumulate in the S-phase. When the drug is eliminated, the affected cells continue to regenerate to the end of cycle. If these cells are not malignant, they enter a quiescent state. It is known that the half life of 5-FU is very short (11 minute). It has not been shown whether 5-FU inihibits the emergence of Ag-NOR proteins, but 5-FU treatment changes the morphology of AgNORs, reducing the number of satellites by aggregation, typically to a single aggregation around nucleoli in a sphenoidal fashion (30,31). Mean Ag-NOR and MI scores of Group B and Group C on days three, five, and seven were higher than Group A except for the results of day one. Group C Ag-NOR values were different from those of Group C: peak Ag-NOR scores were obtained on the third postoperative day and remained higher than the other group’s results, as is also seen in MI values. Hence, as 5-FU is being metabolised, much higher mean values were obtained and these remained high in Group C due to the rebound effect of higher drug dosage. Scores of the cells with a double nucleus were more significant than MI intra-group evaluation and triple evaluation of the first postoperative day was substantially significant (p= 0.0072). Control group values were higher than other groups and again Group B values were higher than Group C values. When the mean values of KC counts and double and triple evaluation of sub-groups were investigated, there were no significant results in Group C. As in the double-nucleus cell, the trend of the counts was the same, i.e. maximum values were obtained from Group A and the lower values from Group C. According to this pattern, somewhat different from Ag-NOR and MI, depressed counts of KC represent the suppression of regeneration for Group C. Other studies have shown that serum bilirubin levels do not change in regenerative events and our results were in accordance with these (16). Alkaline phosphatase (ALP) levels usually increased and this is an important indicator of regeneration if other investigators find that levels of bilirubin do not change as in this study. Alkaline phosphatase levels proportionally increase with the percentage of liver resection (16,17,32,33). Serum ALP levels can function as an indicator of liver function after hepatectomy, but do not reflect morphological regeneration of the liver. Thus, increased ALP levels after hepatectomy may not reflect the cellular proliferation process itself (34). ALT and AST levels were the highest immediately after the resection, then steadily decreased on consecutive days throughout the study, with mean values of 5-FU administered groups being higher than the control group mean values (35-37). In Group B and Group C, we observed significant changes in ALT, AST, ALP, and GGT. Serum GGT profile was modified in a similar way to Ag-NOR results. We observed that aminotransferase levels increased significantly in Group B and C related subgroups as with other parameters. There were relentless changes in serum bilirubin levels with regeneration, which could be 5-FU related. A regenerative response occurs whatever the extent of hepatectomy, but the course of regeneration and expression of growth factors differs according to the volume of resected liver. A better knowledge of these events could improve the clinical results of hepatic resection for primary or metastatic liver disease (38). In the light of these results, it is considered that 5-FU depresses and delays the regeneration of liver in a dose dependent manner through cessation of the cell cycle. If elimination of the drug continues, depressed cell lines resume to proliferate and terminate the programmed cell cycle. |
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| References | |
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