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Effects of 2′-deoxy-5-fluorouridine on regenerating liver following partial hepatectomy in the rat
JOURNAL
OF SURGICAL
45, 181- 186 (1988)
RESEARCH
Effects of 2’-Deoxy-5fluorouridine on Regenerating following Partial Hepatectomy in the Rat
Liver
R. MARK HOYLE, B.S.,’ ALBERTBANES,PH.D., STEPHENBERNARD,M.D., AND COLIN G. THOMAS, JR., M.D. Departments of Surgery and Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina
Submitted for publication January 13, 1987 This study evaluated the effect of 2’deoxy-5-fluorouridine (FUDR) on the regeneration of the liver following partial (68%) hepatectomy in the rat. Male Sprague-Dawley rats weighing between 190 and 240 g underwent partial hepatectomy under ether anesthesia. Twelve hours postoperatively rats received intraperitoneal injections of 2’deoxy-5-fluorouridine or 0.9% NaCl solution as follows: Group I, 0.9% NaCl solution (n = 49); Group II, 89 mg 2’-deoxy-5-fluorouridine/kg ofbody weight (n = 25); and Group III, 178 mg 2’deoxy-5-fluorouridine/kg of body weight (n = 24). Sham groups underwent celiotomy and liver palpation followed by 0.9% NaCl solution injections (n = 5) or low dose 2’-deoxy5-fluorouridine (n = 5) and high dose 2’-deoxy-5-fluorouridine (n = 5). The regenerative ability of the liver was evaluated by weight and deoxyribonucleic acid synthesis in the liver remnant. Result: Both low and high dose 2’deoxy-5-fluorouridine delayed the peak of deoxyribonucleic acid synthesis from 36 to 72 hr as compared to control animals which had maximal synthesis at 25 to 36 hr postoperatively (P < 0.01). Weight of the liver remnants demonstrated a similar pattern. Conclusion: High doses of 2’-deoxy-5-fluorouridine administered intraperitoneally delay, but do not inhibit, liver regeneration following partial (68%) hepatectomy as reflected by DNA synthesis and weight of the remnant. o 1988 Academic
Press, Inc.
INTRODUCTION
Twenty-five percent of patients with colorectal carcinoma will have liver metastasesat time of presentation and two-thirds of patients eventually develop them [I]. Twentyfive percent of this group of patients will be candidates for partial hepatectomy. Fiveyear survival after partial hepatectomy for liver metastases is 25-30%, with recurrent liver disease being the most common cause of death [2]. Adjuvant chemotherapy may be appropriate following partial hepatectomy to treat unresected micrometastases [ 3, 41. 5-Fluorouracil or 2’-deoxy-S-fluoridine (FUDR) have been the most commonly used agents. Whether or not this adjuvant chemotherapy interferes with the ability of the liver to regenerate is not known. r To whom reprint requests should be addressed at Department of Surgery, The University of North Carolina at Chapel Hill, 136 Burnett-Womack Building 2298, Chapel Hill, NC 275 14.
This study was designed to evaluate the effects of FUDR on liver regeneration after partial hepatectomy in the rat. The results were appraised by measuring liver weight and DNA synthesis in the remnant liver of treated and control animals METHODS
Animals and operative procedures. Male Sprague-Dawley rats weighing between 190 and 240 g were caged singly in a room with 12-hr alternating light/dark schedules and allowed a l-week acclimation period after shipment from the supplier (Charles Rivers Co., Wilmington, MA). Rats had free access to laboratory chow (Purina Rat Chow) and water until being fasted 12 hr before the operative procedure. Under ether anesthesia, partial hepatectomy was performed according to the technique of Higgins and Anderson [5]. Sham hepatectomy consisted of celiotomy, mobilization of the liver lobes normally removed at partial hepatectomy and
181
0022-4804/88 $1.50 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form resewed.
182
JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 2, AUGUST 1988 TABLE 1 CATEGORIZATION OF RATS RECEIVING PARTIAL AND SHAM HEPATEC~OMY
Group
n
Operative procedure
mg FUDR/kg body weight in 10 ml 0.9% NaCl solution’
I II III IV V VI
49 25 24 5 5 5
Partial hepatectomyb Partial hepatectomyb Partial hepatectomyb Sham hepatectomyc Sham hepatectomy’ Sham hepatectomy’
89 178 89 178
’ Rats received FUDR and/or 0.9% NaCl solution 12 hr postoperatively. * 68% liver removal. ’ Celiotomy, mobilization, and palpation of liver lobes.
palpation for 8 min, the mean time required for partial hepatectomy. All operations were carried out between 09:30 and 12:30 hr to avoid change in mitotic activity due to diurnal variation [6]. Rats were injected intraperitoneally with 5-FUDR and/or 0.9% NaCl solution 12 hr postoperatively and grouped as shown in Table 1. Three to nine rats from each group were sacrificed at 25,36,48,72, 120, and 180 hr after undergoing partial or sham hepatectomy (Fig. 1, Table 2). Rats from Group I were also sacrificed at 14 and 24 hr. All animals received 0.5 &i [3H]thymidine (ICN, Irving, CA; sp act, 50 Ci/mmole) per gram of body weight, in 1 ml of 0.9% NaCl solution and injected intraperitoneally, 1 hr before sacrifice. Animals were sacrificed by exsanguination under ether anesthesia. At sacrifice, their portal veins were clamped to allow maximal liver exsanguination; their hearts were tapped to collect plasma for radioactive analysis; and their livers were excised, trimmed of fat, and visceral ligaments, blotted, and weighed. Measurement of DNA Synthesis. DNA synthesis was quantitated by determining radioactivity in [3H]thymidine from a perchloric acid extract (PCA). Total DNA was quantitated using a portion of the PCA extract, reaction with diphenylamine [7], and measurement of uv-240 sphectrophotometer
read at 540-nm wavelength. Radioactivity was measured using a Beckmann LS-250 liquid scintillation counter and expressed as cpm/pg DNA in the total sample. Statistical differences for DNA synthesis and liver weight-body weight ratios among groups and time intervals following partial hepatectomy were analyzed by two-way analysis of variance (ANOVA) computer
2600. 2400. 2200. 20001600. 1600. 9 cl 7 % 0
1400. 1200. lOOil. 600. 600. 400. \ 200.
"ep. +SF(IDR 171 . - .mr,W.I .._.. ~ Hep.+E.FUIDR 89 ..
m#nQ
nep.
14 3648 24 A 25
72
120
160
Hours
FIG. 1. Incorporation of [3H]thymidine into DNA following partial or sham hepatectomy in rats receiving FUDR and 0.9% NaCl solution or only 0.9% NaCl solution. Two dosages of FUDR were used: 89 and 178 mg/kg body weight.
183
HOYLE ET AL.: EFFECTS OF FUDR ON LIVER REGENERATION TABLE 2 EFFECTS OF FUDR ON REGENERATION AS MEASURED BY DNA SYNTHESIS“ FOLLOWING PARTIAL OR SHAM HEPATECTOMY IN RATS
Group Time in hoursb 14
24
25
36
12
120
18.39 n=l
-
-
16.90 n=l
-
-
III’
rvf
63.76
-
-
-
-
(18.66) n=7 7 12.32 (506.00) tl=5 1410.62 (454.63) ?l=l 1460.20 (709.54) 403.8 1 (110.19) n=9 769.38 (250.76) n=7 285.65 (100.09) n=3
180
VP
IId
n=l
48
Vg
IC
115.77 (3 1.40) n=4
184.71 (52.8 I) n=3 153.47 (50.80) n=6 161.13 (64.68) n=5 1461.19 (308.74) n=4 337.84 (115.22) n=3 217.35 (50.38) n=3
228.09 (54.95) n=3 140.07 (27.81) n=3 79.22 (15.84) n=6 2575.8 1 (253.70)
-
36.28 n=2
59.25 n=2
95.87 n=l
89.03 n=l
99.02 n=l
32.19 n=l
50.50 n=l
45.70 n=l
73.52 n=l
76.06 n=l
71.46 n=l
n=4
569.54 (61.52) n=3 205.46 (33.38)
-
-
-
-
-
-
n=3
a DNA synthesis measured by incorporation of [3H] thymidine and expressed as mean (SD) countslpg DNA. bTime following partial or sham hepatectomy. ’ Partial hepatectony (n = 49). d Partial hepatectomy + 89 mg FUDR/kg bodyweight (n = 25). ‘Partial hepatectomy + 178 mg FUDR/kg bodyweight (n = 24). ‘Sham hepatectomy (n = 5). g Sham hepatectomy + 89 mg FUDR/kg bodyweight (n = 5). h Sham hepatectomy + 178 mg FUDR/kg bodyweight (n = 5).
programs (Statpak, Walnick Associates, Minneapolis, MN). Differences among and between groups were considered significant if P < 0.05. Statistically significant differences for both DNA synthesis and liver weight to body weight ratios were found (ANOVA, P < 0.001). Then, differences among means of the groups were tested using the StudentNewman-Keuls test (SNK) [8].’ Differences ’ The SNK test incorporates ANOVA data.
among means were considered as significant at P < 0.05 and represent the reported P values. Partial hepatectomy resulted in 68 + 2% of the liver being removed as predetermined with 10 rats in our laboratory, based upon wet weights of excised liver at partial hepatectomy compared to wet weights of remnant liver in animals sacrificed immediately following partial hepatectomy. This figure is consistent with the experience of others using
184
JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 2, AUGUST 1988
the same technique for partial hepatectomy [9]. The amount of liver removed in our study groups was assumed to be 68%. RESULTS
Operative mortality and morbidity. Two rats were lost at operation (n = 113; 1.8% operative mortality). In Group I (partial hepatectomy + 0.9% NaCl solution) and all sham groups, there was no postoperative mortality. In Group II (partial hepatectomy + 89 mg FUDR/kg body weight), one rat died on Day 3 (4% mortality); in Group III (partial hepatectomy + 178 mg FUDR/kg body weight), two rats died 24 hr postoperatively (8% mortality). Drug-treated animals appeared sluggish and looked “sick” as reflected by coarse, ruffled hair until 36 hr postoperatively. All other surviving animals were perceived as being “healthy.” Effects on DNA synthesis. The rates of DNA synthesis for all groups are shown in Fig. 1 and Table 2. For Group I (control animals undergoing partial hepatectomy) DNA synthesis began 14 to 16 hr postoperatively with a primary peak at 25 to 36 hr. In Groups II (partial hepatectomy + 89 mg FUDR/kg body weight) and III (partial hepatectomy + 178 mg FUDR/kg body weight) DNA synthesis was inhibited during the first 48 hr with the primary peak appearing at 72 hr. Group III had a primary peak of DNA synthesis that was twice the magnitude of that for the primary peak of either Group I or Group II (SNK, P < 0.01). The primary peaks had the same magnitude but Group II’s peak was delayed by 36 hr compared to that of Group I (SNK, P < 0.01). Animals undergoing sham hepatectomy with and without drug administration showed no increase in their liver DNA activity at any time. E&cts on liver weight. Liver regeneration based on liver weight remnants is expressed as the liver weight to body weight ratio at sacrifice (Fig. 2, Table 3). Regeneration in the drug-treated groups (Groups II and III) appeared to lag behind that in the control
d 4 b
2.0.
G 1.5.
FIG. 2. Effects of FUDR on regeneration as measured by liver weight/body weight at sacrifice. The only significant difference between control and drug-treated groups was at 72 hr (P < 0.01).
group (Group I) until 120 hr. However, the only statistically significant difference between the groups was at 72 hr (SNK, P < 0.01). Regenerated liver reached the estimated preoperative liver weight between 5 and 7 days postoperatively in both control and drug-treated groups. To account for differences in body weights in the animals as a result of drug effects such as anorexia and gastrointestinal toxicity (data not shown) data on regeneration were expressed as3 Liver wt/body wt (at sacrifice) Estimated liver wt/body wt (preoperatively) Using this method of comparison, again the only difference between Groups I, II, and III was at 72 hr (SNK, P < 0.01). A plot of liver weight at sacrifice (ignoring the effects of changes in body weight) gave comparable results, i.e., the only difference between Group I vs Groups II and III was at 72 hr (SNK, P < 0.01). 3 Estimated liver weight based on 68% removal.
HOYLE ET AL.: EFFECTSOF FUDR ON LIVER REGENERATION TABLE3 EFFECXSOF FUDR ON REGENERATION AS MEASURED BY LIVER WEIGHT/BODY WEIGHT AT SACRIFICE“ Group Time in hoursb
I’
IId
IIP
14
0.0145 (0.00183) ?l=l
-
-
24
0.0157 (0.00193) n=5
-
-
25
0.016 (0.002) n=7
0.015 (0.002)
0.015 (0.001)
n=3
n=3
36
0.018 (0.00 I) Tl=l
0.017
(0.000)
0.016 (0.00 I)
n=6
n=3
48
0.022 (0.001)
0.018 (0.002)
0.020 (0.002)
12
0.028 (0.003)
n=l
n=4
120
0.036 (0.005) n=3
0.035 (0.00 1)
n=3
0.033 (0.000) n=3
0.032 (0.002) n=4
0.030 (0.002)
0.039 (0.009)
n=3
n=3
n=9
180
n=5
n=6
0.02of (0.005)
0.02of (0.001) n=4
’ Mean (SD) liver weight/body weight at sacrifice. b Time following partial or sham hepatectomy. ’ Partial hepatectomy (n = 49).
dPartialhepatectomy + 89mgFUDR/kgbodyweight (n = 25). ePartial hepatectomy+ 178mg FUDR/kg bodyweight(n = 24). ‘Only significant difference among control and drug groups was at 72 hr (P c: 0.01).
DISCUSSION
In selected patients, surgery for hepatic metastasis from colorectal cancer results in a 25-30% Syear survival rate [2]. Although surgery is the primary treatment for such metastasis, adjuvant chemotherapy is sometimes used to treat microscopic metastases that are undetectable at surgery. The high incidence of recurrent disease from pre-
185
sumed micrometastasis after a partial hepatectomy for “localized” liver metastasis raises the question as to whether the results of surgery may be improved with the adjuvant use of intraarterial chemotherapy. Several studies have assessedthe effects of antitumor agents on liver regeneration using the rat model [lo- 151with some drugs inhibiting and some having no observable effects.A few of these studies using the same drug(s) show conflicting data and conclusions based upon parameters chosen to measure liver regeneration. However, the effect of FUDR, one of the more commonly used drugs for hepatic metastasis, on liver regeneration has not been studied. FUDR is converted to FdUMP in vivo which competes with cellular dUMP for the enzyme thymidylate synthetase, blocking the conversion of dUMP to dTMP and ultimately inhibiting DNA synthesis, with some inhibitory effect on RNA synthesis as well. FUDR was given 12hr postoperatively not only to allow the animals adequate time to recover from the effects of surgery and anesthesia, but to allow the drug to act coincident with DNA synthesis. Torres et al. noted that 17.5 + 2.6% of 10 cc 0.9% saline injected intraperitoneally is absorbed from the rat peritoneal cavity in I hr suggesting a prolonged absorption over a few hours [ 161. Giving FUDR 12 hr postoperatively allows the drug to act coincident with DNA synthesis, which in our control animals began at 14 to 16 hr postoperatively with a peak at 25 to 36 hr postoperatively. The dosages used in this study, 89 mg FUDR/kg body weight (Group II) and 178 mg FUDR/kg body weight (Group III) were several times higher than that used in man (0.3 to 0.6 mg FUDR/kg body weight/day for up to 2 weeks). However, these high concentrations did not inhibit DNA synthesis but only delayed the peak synthesis to 72 hr postoperatively. In the control group (I) of partially hepatectomized animals the peak DNA synthesis was at 25 to 36 hr. Group III’s primary peak was twice the magnitude of Group I and II’s peaks, an observation seen in a simi-
186
JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 2, AUGUST 1988
lar study using 5-FU [ 151.This phenomenon is attributed to the fact that normally about 10% of cells in regenerating liver undergo synchronous cell division [ 171. The larger dose, by initially inhibiting DNA synthesis, may have synchronized more cells to divide at a given time. All sham groups had radioactive counts of [3H]thymidine per minute per microgram DNA below 100, and therefore as expected showed no stimulus for regeneration. Plasma from blood drawn from the heart at sacrifice was analyzed for radioactivity to ensure that FUDR did not interfere with the uptake and incorporation of [3H]thymidine into liver cells. Radioactivity was unaffected in all animals, i.e., radioactive counts were similar in all groups for a given amount of plasma analyzed. Data on regeneration based on remnant liver weights in our control group of partially hepatectomized rats are similar to the findings of other investigators [5,9, 181.The only statistically significant difference in regenerative liver weights between control and drugtreated groups was at 72 hr postoperatively with there being no difference at 120 and 180 hr. Our findings indicate that FUDR appeared to only transiently affect the proliferative response of regenerating liver cells. Following partial (3) hepatectomy in the rat, high concentrations of FUDR administered intraperitoneally delay, but do not inhibit liver regeneration as measured by DNA synthesis and remnant liver weights. ACKNOWLEDGMENTS We wish to thank Fernando DiMeo for technical assistance, Dr. Bill Link for assistancewith statistical analysis, and Ann Marie Petri and Doris Eason for preparation of the manuscript. Appreciation is expressed to Roche Laboratories (Division of Hoffmann-LaRoche, Inc., Nutley, NJ) for providing the FUDR. This project was supported in part by the National Cancer Institute (Grant CA17973) and the Gaston County Cancer Society.
REFERENCES 1. Stehlin, J. S., Jr., Hafstrom, L., and Greeff, P. J. Experience with infusion and resection in cancer of the liver. Surg. Gynecol. Obstet. 138: 855, 1974. 2. Meyers, W. C. Neoplasms of the liver. In D. A. Sabiston (Ed.), Textbook ofSurgery. The Biological
Basis of Modern Surgical Practice, 13th ed., Philadelphia: Saunders, 1986. Pp. 1079-1092. 3. Hodges, M. S., Friedland, M., Ahmed, T., Mittelman, A., Berman, H., Katz, S., Morgan, J., and Byrne, D. Treatment of colorectal hepatic metastases by intrahepatic chemotherapy alone or as an adjuvant to complete or partial removal of metastatic disease.Ann. Surg. 203(4): 420, 1986. 4. Kemeny, M. M., Goldbert, D., Beatty, J. D., et ai. Results of a prospective randomized trial of continuous regional chemotherapy and hepatic resection as treatment of hepatic metastases from colorectal primaries. Cancer 57(3): 492, 1986. 5. Higgins, G. M., and Anderson, R. S. Restoration of the liver of the white rat following partial surgical removal. Arch. Surg. 12: 186, 1931. 6. Jaffe, J. J. Diurnal mitotic periodicity in regenerating rat liver. Anat. Rec. 120: 935, 1954. 7. Burton, K. A study of the condition and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biothem. J. 64: 315, 1956. 8. Sokal, R., and Rohlf, F. J. Biometry: The Principles and Practices of Statistics in Biological Research. San Francisco: Freeman, 1969. Pp. 239-246. 9. Brues, A. M., Dnuy, D. R., and Brues, M. C. A quantitative study of cell growth in regenerating liver. Arch. Pathol. 22: 658, 1936. 10. Balazoua, E., Tkacova, E., Kuzela, S., Drobnik, J., and Ujahzy, V. Effect of combined chemotherapy tested in partially hepatectomized rats bearing Zajdela hepatoma. Neoplasma 30(6): 643, 1983. 11. Gavosto, F., and Pileri, A. The effect of administration of 6-mercaptopurine on nucleic acids and alkaline phosphatase of regenerating rat liver. Cancer 11: 222, 1958.
12. Gershbein, L. L., and Pedroso, A. F. Liver regeneration in the presence of antitumor antibiotics and metabolites. Acta Hepato-Gastroenterol. 15: 89, 1968. 13. Nagasue, N., Kobayashi, M., lwaki, A., Kirofumi, Y., Kanashima, R., and Inokughi, K. Effect of 5-fluorouracil on liver regeneration and metabolism after partial hepatectomy in the rat. Cancer 41: 435, 1978.
14. Paschkis, K. E., Bartuska, D., Zayerman, J., Goddard, J. W., and Cantarow, A. Effect of 5-fluorouracil on noncancerous tissue growth. Cancer Res. 19: 1196, 1959. 15. Ryan, C. J., Wood, C. B., and Blumgart, L. H. The effect of 5-lluorouracil on DNA synthesis in the rat liver undergoing compensatory hyperplasia after partial hepatectomy. Clin. Oncol. 9: 43, 1983. 16. Torres, I. J., Litterst, C. L., Guarino, A. M. Transport of model compounds across the peritoneal membrane in the rat. Pharmacology 17: 330, 1978. 17. Bucher, N. L. R. Regeneration of mammalian liver. Int. Rev. Cytol. 15: 245, 1963. 18. Bucher, N. L. R., and McGowan, J. A. Liver regeneration, regulatory mechanisms. In R. Wright et al (Eds.), Liver and biliary disease. Philadelphia: Saunders, 1979. Pp. 2 10-227.
OF SURGICAL
45, 181- 186 (1988)
RESEARCH
Effects of 2’-Deoxy-5fluorouridine on Regenerating following Partial Hepatectomy in the Rat
Liver
R. MARK HOYLE, B.S.,’ ALBERTBANES,PH.D., STEPHENBERNARD,M.D., AND COLIN G. THOMAS, JR., M.D. Departments of Surgery and Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina
Submitted for publication January 13, 1987 This study evaluated the effect of 2’deoxy-5-fluorouridine (FUDR) on the regeneration of the liver following partial (68%) hepatectomy in the rat. Male Sprague-Dawley rats weighing between 190 and 240 g underwent partial hepatectomy under ether anesthesia. Twelve hours postoperatively rats received intraperitoneal injections of 2’deoxy-5-fluorouridine or 0.9% NaCl solution as follows: Group I, 0.9% NaCl solution (n = 49); Group II, 89 mg 2’-deoxy-5-fluorouridine/kg ofbody weight (n = 25); and Group III, 178 mg 2’deoxy-5-fluorouridine/kg of body weight (n = 24). Sham groups underwent celiotomy and liver palpation followed by 0.9% NaCl solution injections (n = 5) or low dose 2’-deoxy5-fluorouridine (n = 5) and high dose 2’-deoxy-5-fluorouridine (n = 5). The regenerative ability of the liver was evaluated by weight and deoxyribonucleic acid synthesis in the liver remnant. Result: Both low and high dose 2’deoxy-5-fluorouridine delayed the peak of deoxyribonucleic acid synthesis from 36 to 72 hr as compared to control animals which had maximal synthesis at 25 to 36 hr postoperatively (P < 0.01). Weight of the liver remnants demonstrated a similar pattern. Conclusion: High doses of 2’-deoxy-5-fluorouridine administered intraperitoneally delay, but do not inhibit, liver regeneration following partial (68%) hepatectomy as reflected by DNA synthesis and weight of the remnant. o 1988 Academic
Press, Inc.
INTRODUCTION
Twenty-five percent of patients with colorectal carcinoma will have liver metastasesat time of presentation and two-thirds of patients eventually develop them [I]. Twentyfive percent of this group of patients will be candidates for partial hepatectomy. Fiveyear survival after partial hepatectomy for liver metastases is 25-30%, with recurrent liver disease being the most common cause of death [2]. Adjuvant chemotherapy may be appropriate following partial hepatectomy to treat unresected micrometastases [ 3, 41. 5-Fluorouracil or 2’-deoxy-S-fluoridine (FUDR) have been the most commonly used agents. Whether or not this adjuvant chemotherapy interferes with the ability of the liver to regenerate is not known. r To whom reprint requests should be addressed at Department of Surgery, The University of North Carolina at Chapel Hill, 136 Burnett-Womack Building 2298, Chapel Hill, NC 275 14.
This study was designed to evaluate the effects of FUDR on liver regeneration after partial hepatectomy in the rat. The results were appraised by measuring liver weight and DNA synthesis in the remnant liver of treated and control animals METHODS
Animals and operative procedures. Male Sprague-Dawley rats weighing between 190 and 240 g were caged singly in a room with 12-hr alternating light/dark schedules and allowed a l-week acclimation period after shipment from the supplier (Charles Rivers Co., Wilmington, MA). Rats had free access to laboratory chow (Purina Rat Chow) and water until being fasted 12 hr before the operative procedure. Under ether anesthesia, partial hepatectomy was performed according to the technique of Higgins and Anderson [5]. Sham hepatectomy consisted of celiotomy, mobilization of the liver lobes normally removed at partial hepatectomy and
181
0022-4804/88 $1.50 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form resewed.
182
JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 2, AUGUST 1988 TABLE 1 CATEGORIZATION OF RATS RECEIVING PARTIAL AND SHAM HEPATEC~OMY
Group
n
Operative procedure
mg FUDR/kg body weight in 10 ml 0.9% NaCl solution’
I II III IV V VI
49 25 24 5 5 5
Partial hepatectomyb Partial hepatectomyb Partial hepatectomyb Sham hepatectomyc Sham hepatectomy’ Sham hepatectomy’
89 178 89 178
’ Rats received FUDR and/or 0.9% NaCl solution 12 hr postoperatively. * 68% liver removal. ’ Celiotomy, mobilization, and palpation of liver lobes.
palpation for 8 min, the mean time required for partial hepatectomy. All operations were carried out between 09:30 and 12:30 hr to avoid change in mitotic activity due to diurnal variation [6]. Rats were injected intraperitoneally with 5-FUDR and/or 0.9% NaCl solution 12 hr postoperatively and grouped as shown in Table 1. Three to nine rats from each group were sacrificed at 25,36,48,72, 120, and 180 hr after undergoing partial or sham hepatectomy (Fig. 1, Table 2). Rats from Group I were also sacrificed at 14 and 24 hr. All animals received 0.5 &i [3H]thymidine (ICN, Irving, CA; sp act, 50 Ci/mmole) per gram of body weight, in 1 ml of 0.9% NaCl solution and injected intraperitoneally, 1 hr before sacrifice. Animals were sacrificed by exsanguination under ether anesthesia. At sacrifice, their portal veins were clamped to allow maximal liver exsanguination; their hearts were tapped to collect plasma for radioactive analysis; and their livers were excised, trimmed of fat, and visceral ligaments, blotted, and weighed. Measurement of DNA Synthesis. DNA synthesis was quantitated by determining radioactivity in [3H]thymidine from a perchloric acid extract (PCA). Total DNA was quantitated using a portion of the PCA extract, reaction with diphenylamine [7], and measurement of uv-240 sphectrophotometer
read at 540-nm wavelength. Radioactivity was measured using a Beckmann LS-250 liquid scintillation counter and expressed as cpm/pg DNA in the total sample. Statistical differences for DNA synthesis and liver weight-body weight ratios among groups and time intervals following partial hepatectomy were analyzed by two-way analysis of variance (ANOVA) computer
2600. 2400. 2200. 20001600. 1600. 9 cl 7 % 0
1400. 1200. lOOil. 600. 600. 400. \ 200.
"ep. +SF(IDR 171 . - .mr,W.I .._.. ~ Hep.+E.FUIDR 89 ..
m#nQ
nep.
14 3648 24 A 25
72
120
160
Hours
FIG. 1. Incorporation of [3H]thymidine into DNA following partial or sham hepatectomy in rats receiving FUDR and 0.9% NaCl solution or only 0.9% NaCl solution. Two dosages of FUDR were used: 89 and 178 mg/kg body weight.
183
HOYLE ET AL.: EFFECTS OF FUDR ON LIVER REGENERATION TABLE 2 EFFECTS OF FUDR ON REGENERATION AS MEASURED BY DNA SYNTHESIS“ FOLLOWING PARTIAL OR SHAM HEPATECTOMY IN RATS
Group Time in hoursb 14
24
25
36
12
120
18.39 n=l
-
-
16.90 n=l
-
-
III’
rvf
63.76
-
-
-
-
(18.66) n=7 7 12.32 (506.00) tl=5 1410.62 (454.63) ?l=l 1460.20 (709.54) 403.8 1 (110.19) n=9 769.38 (250.76) n=7 285.65 (100.09) n=3
180
VP
IId
n=l
48
Vg
IC
115.77 (3 1.40) n=4
184.71 (52.8 I) n=3 153.47 (50.80) n=6 161.13 (64.68) n=5 1461.19 (308.74) n=4 337.84 (115.22) n=3 217.35 (50.38) n=3
228.09 (54.95) n=3 140.07 (27.81) n=3 79.22 (15.84) n=6 2575.8 1 (253.70)
-
36.28 n=2
59.25 n=2
95.87 n=l
89.03 n=l
99.02 n=l
32.19 n=l
50.50 n=l
45.70 n=l
73.52 n=l
76.06 n=l
71.46 n=l
n=4
569.54 (61.52) n=3 205.46 (33.38)
-
-
-
-
-
-
n=3
a DNA synthesis measured by incorporation of [3H] thymidine and expressed as mean (SD) countslpg DNA. bTime following partial or sham hepatectomy. ’ Partial hepatectony (n = 49). d Partial hepatectomy + 89 mg FUDR/kg bodyweight (n = 25). ‘Partial hepatectomy + 178 mg FUDR/kg bodyweight (n = 24). ‘Sham hepatectomy (n = 5). g Sham hepatectomy + 89 mg FUDR/kg bodyweight (n = 5). h Sham hepatectomy + 178 mg FUDR/kg bodyweight (n = 5).
programs (Statpak, Walnick Associates, Minneapolis, MN). Differences among and between groups were considered significant if P < 0.05. Statistically significant differences for both DNA synthesis and liver weight to body weight ratios were found (ANOVA, P < 0.001). Then, differences among means of the groups were tested using the StudentNewman-Keuls test (SNK) [8].’ Differences ’ The SNK test incorporates ANOVA data.
among means were considered as significant at P < 0.05 and represent the reported P values. Partial hepatectomy resulted in 68 + 2% of the liver being removed as predetermined with 10 rats in our laboratory, based upon wet weights of excised liver at partial hepatectomy compared to wet weights of remnant liver in animals sacrificed immediately following partial hepatectomy. This figure is consistent with the experience of others using
184
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the same technique for partial hepatectomy [9]. The amount of liver removed in our study groups was assumed to be 68%. RESULTS
Operative mortality and morbidity. Two rats were lost at operation (n = 113; 1.8% operative mortality). In Group I (partial hepatectomy + 0.9% NaCl solution) and all sham groups, there was no postoperative mortality. In Group II (partial hepatectomy + 89 mg FUDR/kg body weight), one rat died on Day 3 (4% mortality); in Group III (partial hepatectomy + 178 mg FUDR/kg body weight), two rats died 24 hr postoperatively (8% mortality). Drug-treated animals appeared sluggish and looked “sick” as reflected by coarse, ruffled hair until 36 hr postoperatively. All other surviving animals were perceived as being “healthy.” Effects on DNA synthesis. The rates of DNA synthesis for all groups are shown in Fig. 1 and Table 2. For Group I (control animals undergoing partial hepatectomy) DNA synthesis began 14 to 16 hr postoperatively with a primary peak at 25 to 36 hr. In Groups II (partial hepatectomy + 89 mg FUDR/kg body weight) and III (partial hepatectomy + 178 mg FUDR/kg body weight) DNA synthesis was inhibited during the first 48 hr with the primary peak appearing at 72 hr. Group III had a primary peak of DNA synthesis that was twice the magnitude of that for the primary peak of either Group I or Group II (SNK, P < 0.01). The primary peaks had the same magnitude but Group II’s peak was delayed by 36 hr compared to that of Group I (SNK, P < 0.01). Animals undergoing sham hepatectomy with and without drug administration showed no increase in their liver DNA activity at any time. E&cts on liver weight. Liver regeneration based on liver weight remnants is expressed as the liver weight to body weight ratio at sacrifice (Fig. 2, Table 3). Regeneration in the drug-treated groups (Groups II and III) appeared to lag behind that in the control
d 4 b
2.0.
G 1.5.
FIG. 2. Effects of FUDR on regeneration as measured by liver weight/body weight at sacrifice. The only significant difference between control and drug-treated groups was at 72 hr (P < 0.01).
group (Group I) until 120 hr. However, the only statistically significant difference between the groups was at 72 hr (SNK, P < 0.01). Regenerated liver reached the estimated preoperative liver weight between 5 and 7 days postoperatively in both control and drug-treated groups. To account for differences in body weights in the animals as a result of drug effects such as anorexia and gastrointestinal toxicity (data not shown) data on regeneration were expressed as3 Liver wt/body wt (at sacrifice) Estimated liver wt/body wt (preoperatively) Using this method of comparison, again the only difference between Groups I, II, and III was at 72 hr (SNK, P < 0.01). A plot of liver weight at sacrifice (ignoring the effects of changes in body weight) gave comparable results, i.e., the only difference between Group I vs Groups II and III was at 72 hr (SNK, P < 0.01). 3 Estimated liver weight based on 68% removal.
HOYLE ET AL.: EFFECTSOF FUDR ON LIVER REGENERATION TABLE3 EFFECXSOF FUDR ON REGENERATION AS MEASURED BY LIVER WEIGHT/BODY WEIGHT AT SACRIFICE“ Group Time in hoursb
I’
IId
IIP
14
0.0145 (0.00183) ?l=l
-
-
24
0.0157 (0.00193) n=5
-
-
25
0.016 (0.002) n=7
0.015 (0.002)
0.015 (0.001)
n=3
n=3
36
0.018 (0.00 I) Tl=l
0.017
(0.000)
0.016 (0.00 I)
n=6
n=3
48
0.022 (0.001)
0.018 (0.002)
0.020 (0.002)
12
0.028 (0.003)
n=l
n=4
120
0.036 (0.005) n=3
0.035 (0.00 1)
n=3
0.033 (0.000) n=3
0.032 (0.002) n=4
0.030 (0.002)
0.039 (0.009)
n=3
n=3
n=9
180
n=5
n=6
0.02of (0.005)
0.02of (0.001) n=4
’ Mean (SD) liver weight/body weight at sacrifice. b Time following partial or sham hepatectomy. ’ Partial hepatectomy (n = 49).
dPartialhepatectomy + 89mgFUDR/kgbodyweight (n = 25). ePartial hepatectomy+ 178mg FUDR/kg bodyweight(n = 24). ‘Only significant difference among control and drug groups was at 72 hr (P c: 0.01).
DISCUSSION
In selected patients, surgery for hepatic metastasis from colorectal cancer results in a 25-30% Syear survival rate [2]. Although surgery is the primary treatment for such metastasis, adjuvant chemotherapy is sometimes used to treat microscopic metastases that are undetectable at surgery. The high incidence of recurrent disease from pre-
185
sumed micrometastasis after a partial hepatectomy for “localized” liver metastasis raises the question as to whether the results of surgery may be improved with the adjuvant use of intraarterial chemotherapy. Several studies have assessedthe effects of antitumor agents on liver regeneration using the rat model [lo- 151with some drugs inhibiting and some having no observable effects.A few of these studies using the same drug(s) show conflicting data and conclusions based upon parameters chosen to measure liver regeneration. However, the effect of FUDR, one of the more commonly used drugs for hepatic metastasis, on liver regeneration has not been studied. FUDR is converted to FdUMP in vivo which competes with cellular dUMP for the enzyme thymidylate synthetase, blocking the conversion of dUMP to dTMP and ultimately inhibiting DNA synthesis, with some inhibitory effect on RNA synthesis as well. FUDR was given 12hr postoperatively not only to allow the animals adequate time to recover from the effects of surgery and anesthesia, but to allow the drug to act coincident with DNA synthesis. Torres et al. noted that 17.5 + 2.6% of 10 cc 0.9% saline injected intraperitoneally is absorbed from the rat peritoneal cavity in I hr suggesting a prolonged absorption over a few hours [ 161. Giving FUDR 12 hr postoperatively allows the drug to act coincident with DNA synthesis, which in our control animals began at 14 to 16 hr postoperatively with a peak at 25 to 36 hr postoperatively. The dosages used in this study, 89 mg FUDR/kg body weight (Group II) and 178 mg FUDR/kg body weight (Group III) were several times higher than that used in man (0.3 to 0.6 mg FUDR/kg body weight/day for up to 2 weeks). However, these high concentrations did not inhibit DNA synthesis but only delayed the peak synthesis to 72 hr postoperatively. In the control group (I) of partially hepatectomized animals the peak DNA synthesis was at 25 to 36 hr. Group III’s primary peak was twice the magnitude of Group I and II’s peaks, an observation seen in a simi-
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JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 2, AUGUST 1988
lar study using 5-FU [ 151.This phenomenon is attributed to the fact that normally about 10% of cells in regenerating liver undergo synchronous cell division [ 171. The larger dose, by initially inhibiting DNA synthesis, may have synchronized more cells to divide at a given time. All sham groups had radioactive counts of [3H]thymidine per minute per microgram DNA below 100, and therefore as expected showed no stimulus for regeneration. Plasma from blood drawn from the heart at sacrifice was analyzed for radioactivity to ensure that FUDR did not interfere with the uptake and incorporation of [3H]thymidine into liver cells. Radioactivity was unaffected in all animals, i.e., radioactive counts were similar in all groups for a given amount of plasma analyzed. Data on regeneration based on remnant liver weights in our control group of partially hepatectomized rats are similar to the findings of other investigators [5,9, 181.The only statistically significant difference in regenerative liver weights between control and drugtreated groups was at 72 hr postoperatively with there being no difference at 120 and 180 hr. Our findings indicate that FUDR appeared to only transiently affect the proliferative response of regenerating liver cells. Following partial (3) hepatectomy in the rat, high concentrations of FUDR administered intraperitoneally delay, but do not inhibit liver regeneration as measured by DNA synthesis and remnant liver weights. ACKNOWLEDGMENTS We wish to thank Fernando DiMeo for technical assistance, Dr. Bill Link for assistancewith statistical analysis, and Ann Marie Petri and Doris Eason for preparation of the manuscript. Appreciation is expressed to Roche Laboratories (Division of Hoffmann-LaRoche, Inc., Nutley, NJ) for providing the FUDR. This project was supported in part by the National Cancer Institute (Grant CA17973) and the Gaston County Cancer Society.
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