- Email: [email protected]
Effects of arabinosyl cytosine and 5-azacytidine on the intestinal absorption of nutrients
TOXICOLOGY
AND
APPLIED
62,73-80
PHARMACOLOGY
(1982)
Effects of Arabinosyl Cytosine and 5Azacytidine Intestinal Absorption of Nutrients THERESA Department
of Pharmacology
and
Received
Toxicology,
May
4, 1981;
on the
S. CHEN University
accepted
of Louisville,
August
Louisville,
Kentucky
40292
24, 1981
Effects of Arabinosyl Cytosine and S-Azacytidine on the Intestinal Absorption of Nutrients. T. S. (1982). Toxicol. Appl. Pharmacol. 62, 73-80. The purpose of this study was to investigate the effects of two pyrimidine antimetabolites, arabinosyl cytosine (ara-C) and 5 azacytidine (5-aza-C), on the intestinal transport of glucose, amino acids, and electrolytes. Male Swiss-Webster mice were given either 50 mg/kg ara-C or 15 mg/kg 5-aza-C in isotonic saline, ip, once daily for 5 successive days. Using the everted sac technique there was a marked decrease in D-glUCOSe, 3-O-methyl-D-glucose (3MG), and L-tyrosine transport. The rates of glucose, 3MG, and L-tyrosine transport were decreased by 67, 74, and 60%. respectively, in ara-C-treated animals, and 58, 79, and 62%, respectively, in 5-aza-C-treated animals. When intestinal sacs from untreated animals were exposed to ara-C or 5-aza-C ( low3 M) on both mucosal and serosal sides, there was no effect on transport. Furthermore, in animals treated with either ara-C or 5-aza-C there was a significant decrease in transmucosal potential difference and short circuit current associated with decreases in net Na+, Cl-, and HCO; transport across the intestinal mucosa. Simultaneous administration of deoxycytidine, in a two- to threefold molar excess, prevented the impairment of intestinal transport function caused by ara-C, similarly, cytidine prevented the impairment caused by 5-aza-C. The results suggest that active metabolites of ara-C and 5-aza-C are involved in their inhibitory effects on intestinal absorption and that this may be a contributing factor to the malnutrition and diarrhea commonly associated with the administration of these anticancer drugs. CHEN,
Arabinosyl cytosine (ara-C) and 5-azacytidine (5aza-C), in common with many anticancer drugs, can produce undesirable gastrointestinal side effects including anorexia, nausea, weight loss, and diarrhea. Curran et al. ( 1960) demonstrated that the malabsorption syndrome following massive X irradiation of the intestine was due to a direct effect of the irradiation on the absorptive processes of the mucosa. This finding raises the possibility that the malnutrition and diarrhea observed in patients receiving cancer chemotherapeutic agents may also be explainable in part by impairment of intestinal absorption.
The absorptive function of the small intestine is dependent upon a morphologically intact mucosa. Evidence from several lines of investigation indicates that ara-C can produce severe damage to the intestinal crypts of mice (Estensen and Baserga, 1966; Lenaz et al., 1969) and rats (Lenaz and Philips, 1970; Lieberman et al., 1970). Furthermore, Slavin et al. (1978) found that ara-C induced mucosal alterations in the entire gastrointestinal tract when used in the treatment of patients with hematopoietic malignancies. These were characterized by surface and glandular epithelial atypia, immaturity, and necrosis. Whether these mor73
0041-008X/82/010073-08$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
74
THERESA
phological alterations following ara-C treatment are associated with alterations in absorptive function has not been explored. Investigations by Cape1 et al. (1979) indicated that the administration of 5-fluorouracil and methotrexate to rats in doses comparable to the therapeutic dose in man, significantly reduced the intestinal absorption of nutrients. Pretreatment of rats with several anticancer drugs decreased the intestinal absorption of antipyrine (Cape1 et al., 1978). In addition, Sharma and Nagchaudhuri (1976) found that in rats treated with repeated injections of vinblastine for 18 to 24 hr, there was a decrease in the rate of intestinal absorption of L-proline, which was accompanied by marked changes in the morphology of the small intestinal mucosa. The objective of this study was to determine the effects of the antileukemic drugs, ara-C and 5-aza-C, on the intestinal transport of glucose, amino acids, and electrolytes to further elucidate the possible mechanism of gastrointestinal toxicity. Both drugs caused a profound impairment of intestinal transport function which can be prevented by the simultaneous administration of certain endogenous pyrimidine nucleosides. METHODS Materials. Cytosine arabinoside was kindly supplied by the Upjohn Company (Kalamazoo, Mich). 5-Azacytidine, 2’-deoxycytidine, and cytidine were purchased from Sigma Chemical Company (St. Louis, MO.). All radioisotopes were purchased from New England Nuclear Corporation (Boston, Mass.). Animal treatment. Male Swiss-Webster mice (Laboratory Supply Co., Indianapolis, Ind.) of body weight 30 to 40 g were maintained on a 12-hr light-dark cycle in quarters not sprayed with insecticides. Body weights were recorded daily. The animals were given either 50 mg/kg ara-C or 15 mg/kg 5-aza-C in isotonic saline ip once daily for 5 successive days. Control animals received a comparable volume of isotonic saline. In protective studies, 2’-deoxycytidine, 100 mg/kg, and ara-C, 50 mg/kg, in saline were given simultaneously to mice ip for 5 days. Cytidine, 50 mg/kg, and 5-aza-C, 15 mg/ kg, in saline were given together in the same manner. Intestinal transport of sugars and amino acids. The everted intestinal sac technique (Wilson and Wiseman,
S. CHEN 1954) was employed to study the transmucosal transport of glucose, 3-@methyl-D-glucose (3MG), and L-tyrosine. D-[‘4C(U)]Glucose, 3-@[glucose-‘4C(U)]-methylD-glucose, and L-[‘4C(U)]tyrosine were the tracers for analysis. Both mucosal and serosal fluids were composed of mammalian Ringer solution containing 5.5 mM Dglucose, 5 mM 3MG, and 1 mM L-tyrosine. The sacs were incubated at 37” for 1 hr under 1 atm pressure of 95% Gz-5% CO,. At the end of the incubation, the serosal fluid from each sac was removed, centrifuged, and a 50-~1 sample was pipetted into 10 ml of an aqueous scintillation fluid (Kobayashi and Maudsley, 1969) prepared by combining 4 g Omnifluor (98% PPO and 2% Bis-MSB, New England Nuclear Corp.), 1 liter toluene, and 500 ml Triton X-100 (Rohm and Haas, Philadelphia, Pa.). Radioactivity was determined in a liquid scintillation counter, and concentrations were calculated from the specific activities of standard solutions. Electrolyte transport and electrical measurements. The electrical properties of intestine were determined by the Ussing chamber technique (Ussing and Zerahn, 1951). A section of jejunum-ileum was removed and opened along the line of mesenteric attachment, rinsed with mammalian Ringer solution, and mounted in the aperature of a Lucite chamber with an area of 1.3 cm*. Seven milliliters of bathing fluid was placed in each of two chambers separated by the intestinal membrane. The chambers were continuously oxygenated and stirred by bubbling with 95% 02-5% CO*. The entire chamber was kept in a 37” incubator throughout the experiment. Calomel electrodes were used to measure the potential difference (PD) via agar bridges placed adjacent to each side of the membrane. Two agar electrodes were used to short-circuit the membrane and to send current (I) pulses of 20 pA for the measurement of open-circuit resistance (R = PD/I). **Na and 36C1 were used to measure Na+ and Cl- fluxes across the short-circuited membrane. In all experiments, two adjacent jejunal sections of mouse intestine mounted in separate chambers were used to measure simultaneously mucosal-to-serosal (J,,,,,) and serosal-to-mucosal (J,-,) fluxes of Na+ and Cl-. Samples were collected from each chamber at 30min intervals and were counted in an automatic well scintillation counter and a continuous gas flow Geiger counter. The well counter measured the radioactivity of 22Na, while the Geiger counter measured the combined radioactivity of *‘Na and 36C1.Standard solutions of **Na and 36C1were counted simultaneously with the samples and used for calculating the concentrations of ‘*Na and 36C1in each sample. The rates of ion fluxes were calculated according to the equations described by Ussing and Zerahn (1951). Electrolyte fluxes are expressed as microequivalents per hour per square centimeter. Short-circuit current (I,) is expressed as either microequivalents per hour per square centimeter or as microamperes per square centimeter. PD is expressed as millivolts and R as ohms per square centimeter.
CYTOTOXIC
DRUGS ON INTESTINAL TABLE
75
TRANSPORT
1
EFFECTOF ARABINOSYL CYTOSINEAND 5-AZACYTIDINEON INTFSTINAL TRANSPORTOFD-GLUCOSE, ~-O-METHYL-D-GLUCOSE (3MG), AND TYROSINE' D-Glucose Transport rate (rmollglhr)
Treatment Control
% inhibition
14.74 + 1.73 (1lY
Ara-C (50 mg/kg/day, ip X 5 days) Ara-C (IO->
3MG
M)
in vitro
S-Aza-C (lo-’ vitro
M)
in
% inhibition
4.12 t- 0.36 (11)
4.38 + 1.19’ (11)
67
12.63 + 1.23
14
(6) S-Aza-C (15 mg/kg/day, ip X 5 days)
Transport rate (wWg/hr)
L-Tyrosine
58
18.67 + 1.67 (13)
0
% inhibition
2.88 f 0.41 (19)
1.06 f 0.14’ (10)
74
3.29 If: 0.50
20
2.71 k 0.40 (14)
6
79
1.09 f 0.39’
62
0.88 2 0.25’
(8) 4.11 f 0.79 (9)
1.14 f 0.22’
60
(8)
(6)
6.24 + 0.73’ (9)
Transport rate (mWg/hr)
(6) 3
3.45 f 0.23
5
(12)
a Values are R f SE. b Number of animals per group. ’ Significantly different from control (p < 0.01).
RESULTS Glucose and Amino Acid Transport The effects of ara-C and 5-aza-C on the transmucosal transport of glucose, 3MG, and L-tyrosine by everted intestinal sacs are summarized in Table 1. There was a marked decrease in the rate of transport of each compound. The rates of glucose, 3MG, and Ltyrosine transport were decreased by 67, 74, and 60%, respectively, in ara-C-treated animals; and 58, 79, and 62%, respectively, in 5-aza-C-treated animals. When intestinal sacs from untreated animals were exposed to ara-C or 5-aza-C at a concentration of lop3 M on both mucosal and serosal sides, there was no significant effect on transport. Since the 5-day treatment regimen with these two anticancer drugs was associated with a body weight loss of 18.69 k 1.39% (mean -t SE), the effect of fasting on intestinal transport was also studied. A 24-hr
fast resulted in a 16.8 1 + 2.12% weight loss. However, as shown in Table 2, the rates of D-glUCOSC, 3MG, and L-tyrosine transport were markedly increased to 234, 350, and 302% of the control values, respectively. Therefore, the weight loss was not considered to be a contributing factor in the impairment of intestinal absorption observed in animals treated with these drugs. Electrical Parameters Transport
and
Electrolyte
In agreement with our previous studies (Chang et al., 1974), mouse intestine mounted in an Ussing chamber exhibited a PD ranging from 1 to 5 mV, with serosa electropositive to mucosa. The short circuit current ranged from 34 to 42 pA.cm-*. In animals treated with either ara-C or 5-azaC, there was a significant decrease (p < 0.01) in both PD and Z,, across the intestinal mucosa (Table 3). The resistance of the intes-
76
THERESA
S. CHEN
TABLE
2
EFFECTOF FASTING ON INTESTINAL TRANSPORT' D-Glucose
3-O-Methyl-D-glucose
Transport rate (mollglhr)
Treatment Control
% of control
Transport rate (rmollglhr)
14.74 * 1.73 (11Y
24-hr fasting
L-Tyrosine
% of control
4.12 -t 0.36 (11)
34.45 f 1.44’ (10)
14.43 f 1.43’ (8)
234
Transport rate bmol/g/hr)
% of control
2.88 + 0.41 (19) 350
8.70 f 0.34’ (9)
302
a Values are x + SE. b Number of animals per group. ’ Significantly different from control (p i 0.01).
tinal membrane was slightly increased in drug-treated animals but statistically was not significantly different from untreated controls. The results of simultaneous measurements of Na+ and Cl- fluxes in control and treated animals are summarized in Table 4. In controls, the net Na+ and Cl fluxes were 1-3.04 and -4.03 peq . hr-’ . cm-2, respectively (+ indicates net M - S and - indicates net S - M flux). These values confirm our previous findings that in mouse small intestine there is simultaneous active Na+ absorption and active Cl- secretion which are hydrogen carbonate dependent (Chang et al., 1974). The observed Z,, in control mouse intestine was small, being less than the sum of .Zz
and Jz& and yielding a negative net residual flux (Jte,.) The negative net residual flux is attributable to hydrogen carbonate absorption as demonstrated previously (Chang et al., 1974). The administration of anticancer drugs induced a significant decrease in Jf;it, with no significant change in Jz,,,, resulting in a marked decrease in net Na+ absorption from a control value of +3.04 to +0.40 and +0.98 peq - hr-’ . cm-‘, respectively, in ara-C- and 5-aza-C-treated animals. Likewise, there was a significant decrease in Jf!.,,, with no significant change in JC’m-s, resulting in a marked decrease in net Cl- secretion in ara-C-treated mice. In 5aza-C-treated animals, small increases in JC’,,,+s and decreases in Jz,,, resulted in a
TABLE
3
EFFECTOF ARABINOSYL CYTOSINEAND 5-AZACYTIDINEON ELECTRICALPARAMETERS' Treatment
rib
PD (mV)
R (B.cm’)
I, (pA.cm-‘)
Control Ara-C (50 mg/kg/day, ip x 5 days) 5-Aza-C (15 mg/kg/day, ip X 5 days)
11
4.05 k 0.13
79.95 i 6.27
45.58 t 2.22
7
1.43 + 0.22
107.16 k 10.09
13.29 + 1.45’
6
0.64 + 0.10
90.57 f 6.64
4.94 f 0.87’
r?Values are R + SE. b Number of animals per group. c Significantly different from control (p < 0.01).
CYTOTOXIC
DRUGS ON INTESTINAL TABLE
EFFECT
OF ARABINOSYL
CYTOSINE
AND
4
5-AZACYTIDINE
Ion
J m-s
Control (1l)d
Na*’ CF
11.48 + 0.60 7.32 + 0.98
8.90 + 0.64 +3.04 + 0.62 10.85 + 1.06 -4.03 f 0.04
Ara-C (7)
Naz2 CS6
8.77 + 0.57’ 5.37 f 0.59
8.02 f 0.29 +0.40 f 0.10’ 5.77 + 0.42’ -0.89 + 0.22’
5-Aza-C
NaZ2 CP
8.75 + 0.52’ 8.79 + 1.76
8.57 k 0.62 9.02 + 0.86
(6)
ON ELECTROLYTE
Percentage inhibitionb
Treatment
J s-ml
77
TRANSPORT
J net
+.098 f 0.26’ -0.81 + 0.16
TRANSPORT’
I SE 1.70 + 0.08
Et
Percentage inhibitionb
-5.37
f 0.58
82
0.50 + 0.05’ -0.79
f 0.27’
85
75
0.18 f 0.03’ -1.61
f 0.39’
70
a Values are R 2 SE, expressed as peq. h-’ . cm-‘. ’ Percentage decrease in J.., and &. ‘J.“,, = L - (Et - J:;,). d Number of animals per group. ’ Significantly different from control (p < 0.01).
significant decrease in net Cl- secretion. Therefore, the results clearly demonstrated an overall inhibition of both net Na+ and net Cl- fluxes across the small intestine of araC- and 5-aza-C-treated mice. Furthermore, the hydrogen carbonate flux (calculated as J,“ct) was also significantly reduced from a control value of -5.37 to -0.79 and -1.61 peq * hr-’ - cm-*, respectively, in ara-C- and 5-aza-C-treated mice. Protective Effects of 2-Deoxycytidine and Cytidine on Ara-C- and 5-Aza-C-Induced Inhibition of Transport Mice were simultaneously given ara-C and 2’-deoxycytidine ip for 5 days and the rate of 3MG transport by everted intestinal sacs was determined. As shown in Table 5, the rate of 3MG transport was reduced to 15% of the control values in animals treated with ara-C alone. However, there was no significant effect on 3MG transport in animals receiving 2’-deoxycytidine and ara-C simultaneously. In addition, the body weight loss induced by ara-C treatment was abolished by the concurrent administration of 2’-
deoxycytidine. 2’-Deoxycytidine alone had no effect on 3MG transport. Likewise, 5aza-C alone reduced the rate of 3MG transport to only 12% of the control values. Simultaneous administration of cytidine in a dose of 50 mg/kg, ip, which is three to four times greater than that of 5-aza-C, prevented the inhibition of 3MG transport and partially prevented the body weight loss caused by 5-aza-C. Cytidine alone had no effect on 3MG transport. DISCUSSION It has been reported tha ara-C, administrated ip to mice in the optimum therapeutic dosage schedule of 15 g/kg/dose, every 3 hr for 3 days, resulted in progressive damage to the crypt epithelial cells of the intestinal mucosa (Leach et al., 1969). Results of the present study demonstrated that ara-C and 5-aza-C, administered in doses comparable to those used clinically, caused generalized impairment of active intestinal transport processes in mice. Both drugs caused profound depression of glucose, amino acid, and Na absorption (associated with decreases in
78
THERESA
S. CHEN
TABLE PROTECTIVE
EFFECTOF
5
2'-DEOXYCYTIDINEAND CYTIDINEON ANTICANCER 3-O-METHYL-D-GLUCOSE (3MG) TRANSPORT“
Treatment
n*
Control 2’-Deoxycytidine Ara-C 2’-Deoxycytidine + Ara-C Cytidine 5-Aza-C Cytidine + 5-Aza-C
I 6 10 9 5 8 6
a Values are x + SE. b Number of animals per group. ’ Change from initial; + indicates d Not significantly different from
3MG transport (wol/g/hr)
weight control.
rate
7.13 6.38 1.06 5.88 6.12 0.88 5.88
+ iz t + f + +
gain,
- indicates
p value
0.68 0.71 0.14 0.63 0.50 0.25 0.50
the transmucosal PD and I,,), as well as a substantial weight loss. This functional impairment was due to a direct toxic effect of the drugs rather than a secondary effect of weight loss, since comparable weight loss caused by fasting actually resulted in significant enhancement of glucose and amino acid absorption. The latter observation is in agreement with the finding of Kershaw et al. ( 1960) that starvation resulted in an enhancement of nutrient absorption in the rat. The anticancer activity of ara-C and 5aza-C is dependent upon phosphorylation in the body with formation of the presumed active cytotoxic species, ara-CTP, and 5aza-CTP (Kessel et al., 1967; Li et al., 1970). The observed lack of effect on intestinal transport function during short-term in vitro exposure suggested that metabolic activation was also involved in gastrointestinal toxicity. This lack of toxicity in vitro was probably the result of insufficient exposure time but could also occur if activation at a remote site was required for the toxic effects on the gastrointestinal tract. However, this is unlikely since the active nucleotides are large ionic compounds which are mainly restricted to an intracellular location (Sadee
NSd co.01 NS NS
weight
DRUGS
INDUCED
Body weight change’ (o/o) +0.98 +I.62 -11.83 +2.03 -0.39 -20.68 -8.15
f + + f -c f +_
0.29 0.87 1.23 0.63 1.66 1.22 2.24
INHIBITIONOF
p value
NS
loss.
and Wong, 1977). Furthermore, preliminary studies in this investigation revealed that treatment for only 1 to 2 days caused no impairment of transport function, suggesting that both the time of exposure and drug concentration are important determinants of toxic effects (Mellett, 1974). Since the time course of morphologic changes (Leach et al., 1969) and functional changes induced by ara-C are similar it is possible that the impairment of intestinal function is a consequence of morphological alterations. The observed gastrointestinal toxicity was largely preventable by pyrimidine nucleosides capable of competitively inhibiting the formation of the active nucleotides of ara-C and 5-aza-C. Lee et al. (1975) demonstrated that in fibrosarcoma and leukemic cells, deoxycytidine and cytidine were potent competitive inhibitors of the phosphorylation of ara-C and 5-aza-C, respectively. It has been shown that simultaneous oral administration of 2’-deoxycytidine in doses two to three times as high as those of ara-C prevented drug-related lethal toxicity in mice yet the antitumor activity of ara-C remained (Buchman et al., 1979). Evans and Hanka (1968) showed that the antileukemic activity of 5-
CYTOTOXIC
DRUGS
ON
azacytidine could be reveresed in vivo by either cytidine or uridine. Similarly, the present study showed that the suppression of glucose absorption and body weight loss caused by ara-C was completely prevented by simultaneous treatment with 2’-deoxycytidine, and the suppression of glucose absorption caused by 5-aza-C was largely prevented by simultaneous administration of cytidine, even though the body weight loss was only partially prevented. Malnutrition is a common clinical finding in cancer patients. While this may be due in part to poorly understood systemic effects of malignant neoplasms, the present investigation demonstrated that anticancer drugs are also capable of causing or aggrevating the condition by depression of intestinal absorptive activity. Furthermore, the protective effects of 2’-deoxycytidine and cytidine on ara-C- and 5-aza-C-induced inhibition of glucose absorption may be beneficial in preventing the related gastrointestinal toxicity by anticancer drugs. ACKNOWLEDGMENTS The author is indebted to Dr. K. C. Huang for his valuable discussion and review of this manuscript and to Dr. P. W. O’Connell of the Upjohn Company for the arabinosyl cytosine. This study was supported in part by American Cancer Society Grant In 1llE and USPHS Grant CA-25252-02.
REFERENCES BUCHMAN, V. M., BELYANCHIKOVA, N. I., MKHEIDZE, D. M., LITOVCHENKO, T. A., LICHINITSER, M. R., BARKHOTKINA, M. F. AND SVET MOLDAVSKY. G. J. (1979). 2’-Deoxycytidine hydrochloride protection of mice against the lethal toxicity of cytosine arabinoside. Cancer Chemother. Pharmacol. 3, 229-234. CAPEL, I. D., JENNER, M., PINNOCK, M. H., ANDWILLIAMS, D. C. (1978). The effect of anti-cancer drugs on the plasma disposition of antipyrine and the biliary excretion of phenolphthalein in the rat. B&hem. Pharmacol. 27, 1413-1416. CAPEL, I. D., PINNOCK, M. H., AND WILLIAMS, D. C. (1979). An in vitro assessment of the effect of cyto-
INTESTINAL
TRANSPORT
79
toxic drugs upon the intestinal absorption of nutrients in rats. Eur. J. Cancer 15, 127-l 31. CHANG, L. R., CHEN, T. S. T., AND HUANG, K. C. (1974). Electrolyte transport across the mouse small intestine. Proc. Sot. Exp. Biol. Med. 145, 1220-1224. CURRAN, P. F., WEBSTER, E. W., AND HOVESPIAN, J. A, (1960). The effect of X-irradiation on sodium and water transport in rat ileum. Radiat. Res. 13, 369-380. ESTENSEN, R. D., AND BASERGA, R. (1966). Puromycin induced necrosis of crypt cells of the small intestine of mouse. J. Cell Biol. 30, 13-22. EVANS, J. S., AND HANKA, L. J. (1968). The in vivo activity of combinations of S-azacytidine and cytidine on leukemia L-1210. Experientia 24, 922-923. KERSHAW, T. G., NEAME, K. D., AND WISEMAN, G. (1960). The effect of semistarvation on absorption by the rat small intestine in vitro and in vivo. J. Physiol. 152, 182-190. KESSEL, D., HALL, T. C., AND WODINSKY, I. (1967). Transport and phosphorylation as factors in the antitumor action of cytosine arabinoside. Science 156, 1240-1241. KOBAYASHI, Y., AND MAUDSLEY, D. V. (1969). Practical aspects of liquid scintillation counting. Methods Biochem. Anal. 17, 55-133. LEACH, W. B., LASTER, W. R., JR., MAYO, J. G., GRISWOLD, D. P., JR., AND SCHABEL, F. M., JR. (1969). Toxicity studies in mice treated with 1-@-D-arabinofuranosylcytosine (ara-C). Cancer Res. 29, 529535. LEE, T., KARON, M., AND MOMPARLER, R. L. (1975). Cellular phosphorylation of l-P-D-arabinofuranosylcytosine and .5-azacytidine with intact fibrosarcoma and leukemic cells. Cancer Res. 35, 2506-2510. LENAZ, L., AND PHILIPS, F. S. (1970). Effects of arabinosylcytosine nucleosides on DNA synthesis in rats. Cancer Res. 30, 1961-1962. LENAZ, L., STERNBERG, S. S., AND PHILIPS, F. S. (1969). Cytotoxic effects of I-B-D-arabinofuranosyl5-fluorocytosine and of l+D-arabinofuranosylcytosine in proliferating tissues in mice. Cancer Res. 29, 1790-1798. LI, L. H., OLIN, E. J., BUSKIRK, H. H., AND REINEKE, L. M. (1970). Cytotoxicity and mode of action of 5azacytidine on Ll210 leukemia. Cancer Res. 30, 2760-2769. LIEBERMAN, M. W., VERBIN, R. S., LANDAY, M., LIANG, H., FARBER, E., LEE, T. N., AND STARR, R. (1970). A probable role for protein synthesis in intestinal epithelial cell damage induced in vivo by cytosine arabinoside, nitrogen mustard, or X-irradiation. Cancer Res. 30, 942-951. MELLETT, L. B. (1974). The constancy of the product of concentration and time. In Antineoplastic and Immunosuppressive Agents (A. C. Sartorelli and D. G.
80
THERESA
Johns, eds.), Part I, pp. 330-340. Springer-Verlag. Berlin. SADEE, W., AND WONG, C. G. (1977). Pharmacokinetics of 5-fluorouracil: Interrelationship with biochemical kinetics in monitoring therapy. Clin. Pharmacokinet. 2, 437-450. SHARMA, R. K., AND NAGCHAUDHURI, J. (1976). A study of amino acid absorption from small intestines of vinblastin treated rats with altered mucosal morphology. Indian J. Med. Rex 64, 1225-1234. SLAVIN, R. E., DIAS, M. A., AND SARAL, R. (1978).
S. CHEN Cytosine arabinoside induced gastrointestinal toxic alterations in sequential chemotherapeutic protocols. Cancer 42, 1747-1759. USSING, H. H., AND ZERAHN, K. (195 1). Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol. &and. 23, 110-127. WILSON, T. H., AND WISEMAN. G. (1954). The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J. Physiol. 123, 116- 125.
AND
APPLIED
62,73-80
PHARMACOLOGY
(1982)
Effects of Arabinosyl Cytosine and 5Azacytidine Intestinal Absorption of Nutrients THERESA Department
of Pharmacology
and
Received
Toxicology,
May
4, 1981;
on the
S. CHEN University
accepted
of Louisville,
August
Louisville,
Kentucky
40292
24, 1981
Effects of Arabinosyl Cytosine and S-Azacytidine on the Intestinal Absorption of Nutrients. T. S. (1982). Toxicol. Appl. Pharmacol. 62, 73-80. The purpose of this study was to investigate the effects of two pyrimidine antimetabolites, arabinosyl cytosine (ara-C) and 5 azacytidine (5-aza-C), on the intestinal transport of glucose, amino acids, and electrolytes. Male Swiss-Webster mice were given either 50 mg/kg ara-C or 15 mg/kg 5-aza-C in isotonic saline, ip, once daily for 5 successive days. Using the everted sac technique there was a marked decrease in D-glUCOSe, 3-O-methyl-D-glucose (3MG), and L-tyrosine transport. The rates of glucose, 3MG, and L-tyrosine transport were decreased by 67, 74, and 60%. respectively, in ara-C-treated animals, and 58, 79, and 62%, respectively, in 5-aza-C-treated animals. When intestinal sacs from untreated animals were exposed to ara-C or 5-aza-C ( low3 M) on both mucosal and serosal sides, there was no effect on transport. Furthermore, in animals treated with either ara-C or 5-aza-C there was a significant decrease in transmucosal potential difference and short circuit current associated with decreases in net Na+, Cl-, and HCO; transport across the intestinal mucosa. Simultaneous administration of deoxycytidine, in a two- to threefold molar excess, prevented the impairment of intestinal transport function caused by ara-C, similarly, cytidine prevented the impairment caused by 5-aza-C. The results suggest that active metabolites of ara-C and 5-aza-C are involved in their inhibitory effects on intestinal absorption and that this may be a contributing factor to the malnutrition and diarrhea commonly associated with the administration of these anticancer drugs. CHEN,
Arabinosyl cytosine (ara-C) and 5-azacytidine (5aza-C), in common with many anticancer drugs, can produce undesirable gastrointestinal side effects including anorexia, nausea, weight loss, and diarrhea. Curran et al. ( 1960) demonstrated that the malabsorption syndrome following massive X irradiation of the intestine was due to a direct effect of the irradiation on the absorptive processes of the mucosa. This finding raises the possibility that the malnutrition and diarrhea observed in patients receiving cancer chemotherapeutic agents may also be explainable in part by impairment of intestinal absorption.
The absorptive function of the small intestine is dependent upon a morphologically intact mucosa. Evidence from several lines of investigation indicates that ara-C can produce severe damage to the intestinal crypts of mice (Estensen and Baserga, 1966; Lenaz et al., 1969) and rats (Lenaz and Philips, 1970; Lieberman et al., 1970). Furthermore, Slavin et al. (1978) found that ara-C induced mucosal alterations in the entire gastrointestinal tract when used in the treatment of patients with hematopoietic malignancies. These were characterized by surface and glandular epithelial atypia, immaturity, and necrosis. Whether these mor73
0041-008X/82/010073-08$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
74
THERESA
phological alterations following ara-C treatment are associated with alterations in absorptive function has not been explored. Investigations by Cape1 et al. (1979) indicated that the administration of 5-fluorouracil and methotrexate to rats in doses comparable to the therapeutic dose in man, significantly reduced the intestinal absorption of nutrients. Pretreatment of rats with several anticancer drugs decreased the intestinal absorption of antipyrine (Cape1 et al., 1978). In addition, Sharma and Nagchaudhuri (1976) found that in rats treated with repeated injections of vinblastine for 18 to 24 hr, there was a decrease in the rate of intestinal absorption of L-proline, which was accompanied by marked changes in the morphology of the small intestinal mucosa. The objective of this study was to determine the effects of the antileukemic drugs, ara-C and 5-aza-C, on the intestinal transport of glucose, amino acids, and electrolytes to further elucidate the possible mechanism of gastrointestinal toxicity. Both drugs caused a profound impairment of intestinal transport function which can be prevented by the simultaneous administration of certain endogenous pyrimidine nucleosides. METHODS Materials. Cytosine arabinoside was kindly supplied by the Upjohn Company (Kalamazoo, Mich). 5-Azacytidine, 2’-deoxycytidine, and cytidine were purchased from Sigma Chemical Company (St. Louis, MO.). All radioisotopes were purchased from New England Nuclear Corporation (Boston, Mass.). Animal treatment. Male Swiss-Webster mice (Laboratory Supply Co., Indianapolis, Ind.) of body weight 30 to 40 g were maintained on a 12-hr light-dark cycle in quarters not sprayed with insecticides. Body weights were recorded daily. The animals were given either 50 mg/kg ara-C or 15 mg/kg 5-aza-C in isotonic saline ip once daily for 5 successive days. Control animals received a comparable volume of isotonic saline. In protective studies, 2’-deoxycytidine, 100 mg/kg, and ara-C, 50 mg/kg, in saline were given simultaneously to mice ip for 5 days. Cytidine, 50 mg/kg, and 5-aza-C, 15 mg/ kg, in saline were given together in the same manner. Intestinal transport of sugars and amino acids. The everted intestinal sac technique (Wilson and Wiseman,
S. CHEN 1954) was employed to study the transmucosal transport of glucose, 3-@methyl-D-glucose (3MG), and L-tyrosine. D-[‘4C(U)]Glucose, 3-@[glucose-‘4C(U)]-methylD-glucose, and L-[‘4C(U)]tyrosine were the tracers for analysis. Both mucosal and serosal fluids were composed of mammalian Ringer solution containing 5.5 mM Dglucose, 5 mM 3MG, and 1 mM L-tyrosine. The sacs were incubated at 37” for 1 hr under 1 atm pressure of 95% Gz-5% CO,. At the end of the incubation, the serosal fluid from each sac was removed, centrifuged, and a 50-~1 sample was pipetted into 10 ml of an aqueous scintillation fluid (Kobayashi and Maudsley, 1969) prepared by combining 4 g Omnifluor (98% PPO and 2% Bis-MSB, New England Nuclear Corp.), 1 liter toluene, and 500 ml Triton X-100 (Rohm and Haas, Philadelphia, Pa.). Radioactivity was determined in a liquid scintillation counter, and concentrations were calculated from the specific activities of standard solutions. Electrolyte transport and electrical measurements. The electrical properties of intestine were determined by the Ussing chamber technique (Ussing and Zerahn, 1951). A section of jejunum-ileum was removed and opened along the line of mesenteric attachment, rinsed with mammalian Ringer solution, and mounted in the aperature of a Lucite chamber with an area of 1.3 cm*. Seven milliliters of bathing fluid was placed in each of two chambers separated by the intestinal membrane. The chambers were continuously oxygenated and stirred by bubbling with 95% 02-5% CO*. The entire chamber was kept in a 37” incubator throughout the experiment. Calomel electrodes were used to measure the potential difference (PD) via agar bridges placed adjacent to each side of the membrane. Two agar electrodes were used to short-circuit the membrane and to send current (I) pulses of 20 pA for the measurement of open-circuit resistance (R = PD/I). **Na and 36C1 were used to measure Na+ and Cl- fluxes across the short-circuited membrane. In all experiments, two adjacent jejunal sections of mouse intestine mounted in separate chambers were used to measure simultaneously mucosal-to-serosal (J,,,,,) and serosal-to-mucosal (J,-,) fluxes of Na+ and Cl-. Samples were collected from each chamber at 30min intervals and were counted in an automatic well scintillation counter and a continuous gas flow Geiger counter. The well counter measured the radioactivity of 22Na, while the Geiger counter measured the combined radioactivity of *‘Na and 36C1.Standard solutions of **Na and 36C1were counted simultaneously with the samples and used for calculating the concentrations of ‘*Na and 36C1in each sample. The rates of ion fluxes were calculated according to the equations described by Ussing and Zerahn (1951). Electrolyte fluxes are expressed as microequivalents per hour per square centimeter. Short-circuit current (I,) is expressed as either microequivalents per hour per square centimeter or as microamperes per square centimeter. PD is expressed as millivolts and R as ohms per square centimeter.
CYTOTOXIC
DRUGS ON INTESTINAL TABLE
75
TRANSPORT
1
EFFECTOF ARABINOSYL CYTOSINEAND 5-AZACYTIDINEON INTFSTINAL TRANSPORTOFD-GLUCOSE, ~-O-METHYL-D-GLUCOSE (3MG), AND TYROSINE' D-Glucose Transport rate (rmollglhr)
Treatment Control
% inhibition
14.74 + 1.73 (1lY
Ara-C (50 mg/kg/day, ip X 5 days) Ara-C (IO->
3MG
M)
in vitro
S-Aza-C (lo-’ vitro
M)
in
% inhibition
4.12 t- 0.36 (11)
4.38 + 1.19’ (11)
67
12.63 + 1.23
14
(6) S-Aza-C (15 mg/kg/day, ip X 5 days)
Transport rate (wWg/hr)
L-Tyrosine
58
18.67 + 1.67 (13)
0
% inhibition
2.88 f 0.41 (19)
1.06 f 0.14’ (10)
74
3.29 If: 0.50
20
2.71 k 0.40 (14)
6
79
1.09 f 0.39’
62
0.88 2 0.25’
(8) 4.11 f 0.79 (9)
1.14 f 0.22’
60
(8)
(6)
6.24 + 0.73’ (9)
Transport rate (mWg/hr)
(6) 3
3.45 f 0.23
5
(12)
a Values are R f SE. b Number of animals per group. ’ Significantly different from control (p < 0.01).
RESULTS Glucose and Amino Acid Transport The effects of ara-C and 5-aza-C on the transmucosal transport of glucose, 3MG, and L-tyrosine by everted intestinal sacs are summarized in Table 1. There was a marked decrease in the rate of transport of each compound. The rates of glucose, 3MG, and Ltyrosine transport were decreased by 67, 74, and 60%, respectively, in ara-C-treated animals; and 58, 79, and 62%, respectively, in 5-aza-C-treated animals. When intestinal sacs from untreated animals were exposed to ara-C or 5-aza-C at a concentration of lop3 M on both mucosal and serosal sides, there was no significant effect on transport. Since the 5-day treatment regimen with these two anticancer drugs was associated with a body weight loss of 18.69 k 1.39% (mean -t SE), the effect of fasting on intestinal transport was also studied. A 24-hr
fast resulted in a 16.8 1 + 2.12% weight loss. However, as shown in Table 2, the rates of D-glUCOSC, 3MG, and L-tyrosine transport were markedly increased to 234, 350, and 302% of the control values, respectively. Therefore, the weight loss was not considered to be a contributing factor in the impairment of intestinal absorption observed in animals treated with these drugs. Electrical Parameters Transport
and
Electrolyte
In agreement with our previous studies (Chang et al., 1974), mouse intestine mounted in an Ussing chamber exhibited a PD ranging from 1 to 5 mV, with serosa electropositive to mucosa. The short circuit current ranged from 34 to 42 pA.cm-*. In animals treated with either ara-C or 5-azaC, there was a significant decrease (p < 0.01) in both PD and Z,, across the intestinal mucosa (Table 3). The resistance of the intes-
76
THERESA
S. CHEN
TABLE
2
EFFECTOF FASTING ON INTESTINAL TRANSPORT' D-Glucose
3-O-Methyl-D-glucose
Transport rate (mollglhr)
Treatment Control
% of control
Transport rate (rmollglhr)
14.74 * 1.73 (11Y
24-hr fasting
L-Tyrosine
% of control
4.12 -t 0.36 (11)
34.45 f 1.44’ (10)
14.43 f 1.43’ (8)
234
Transport rate bmol/g/hr)
% of control
2.88 + 0.41 (19) 350
8.70 f 0.34’ (9)
302
a Values are x + SE. b Number of animals per group. ’ Significantly different from control (p i 0.01).
tinal membrane was slightly increased in drug-treated animals but statistically was not significantly different from untreated controls. The results of simultaneous measurements of Na+ and Cl- fluxes in control and treated animals are summarized in Table 4. In controls, the net Na+ and Cl fluxes were 1-3.04 and -4.03 peq . hr-’ . cm-2, respectively (+ indicates net M - S and - indicates net S - M flux). These values confirm our previous findings that in mouse small intestine there is simultaneous active Na+ absorption and active Cl- secretion which are hydrogen carbonate dependent (Chang et al., 1974). The observed Z,, in control mouse intestine was small, being less than the sum of .Zz
and Jz& and yielding a negative net residual flux (Jte,.) The negative net residual flux is attributable to hydrogen carbonate absorption as demonstrated previously (Chang et al., 1974). The administration of anticancer drugs induced a significant decrease in Jf;it, with no significant change in Jz,,,, resulting in a marked decrease in net Na+ absorption from a control value of +3.04 to +0.40 and +0.98 peq - hr-’ . cm-‘, respectively, in ara-C- and 5-aza-C-treated animals. Likewise, there was a significant decrease in Jf!.,,, with no significant change in JC’m-s, resulting in a marked decrease in net Cl- secretion in ara-C-treated mice. In 5aza-C-treated animals, small increases in JC’,,,+s and decreases in Jz,,, resulted in a
TABLE
3
EFFECTOF ARABINOSYL CYTOSINEAND 5-AZACYTIDINEON ELECTRICALPARAMETERS' Treatment
rib
PD (mV)
R (B.cm’)
I, (pA.cm-‘)
Control Ara-C (50 mg/kg/day, ip x 5 days) 5-Aza-C (15 mg/kg/day, ip X 5 days)
11
4.05 k 0.13
79.95 i 6.27
45.58 t 2.22
7
1.43 + 0.22
107.16 k 10.09
13.29 + 1.45’
6
0.64 + 0.10
90.57 f 6.64
4.94 f 0.87’
r?Values are R + SE. b Number of animals per group. c Significantly different from control (p < 0.01).
CYTOTOXIC
DRUGS ON INTESTINAL TABLE
EFFECT
OF ARABINOSYL
CYTOSINE
AND
4
5-AZACYTIDINE
Ion
J m-s
Control (1l)d
Na*’ CF
11.48 + 0.60 7.32 + 0.98
8.90 + 0.64 +3.04 + 0.62 10.85 + 1.06 -4.03 f 0.04
Ara-C (7)
Naz2 CS6
8.77 + 0.57’ 5.37 f 0.59
8.02 f 0.29 +0.40 f 0.10’ 5.77 + 0.42’ -0.89 + 0.22’
5-Aza-C
NaZ2 CP
8.75 + 0.52’ 8.79 + 1.76
8.57 k 0.62 9.02 + 0.86
(6)
ON ELECTROLYTE
Percentage inhibitionb
Treatment
J s-ml
77
TRANSPORT
J net
+.098 f 0.26’ -0.81 + 0.16
TRANSPORT’
I SE 1.70 + 0.08
Et
Percentage inhibitionb
-5.37
f 0.58
82
0.50 + 0.05’ -0.79
f 0.27’
85
75
0.18 f 0.03’ -1.61
f 0.39’
70
a Values are R 2 SE, expressed as peq. h-’ . cm-‘. ’ Percentage decrease in J.., and &. ‘J.“,, = L - (Et - J:;,). d Number of animals per group. ’ Significantly different from control (p < 0.01).
significant decrease in net Cl- secretion. Therefore, the results clearly demonstrated an overall inhibition of both net Na+ and net Cl- fluxes across the small intestine of araC- and 5-aza-C-treated mice. Furthermore, the hydrogen carbonate flux (calculated as J,“ct) was also significantly reduced from a control value of -5.37 to -0.79 and -1.61 peq * hr-’ - cm-*, respectively, in ara-C- and 5-aza-C-treated mice. Protective Effects of 2-Deoxycytidine and Cytidine on Ara-C- and 5-Aza-C-Induced Inhibition of Transport Mice were simultaneously given ara-C and 2’-deoxycytidine ip for 5 days and the rate of 3MG transport by everted intestinal sacs was determined. As shown in Table 5, the rate of 3MG transport was reduced to 15% of the control values in animals treated with ara-C alone. However, there was no significant effect on 3MG transport in animals receiving 2’-deoxycytidine and ara-C simultaneously. In addition, the body weight loss induced by ara-C treatment was abolished by the concurrent administration of 2’-
deoxycytidine. 2’-Deoxycytidine alone had no effect on 3MG transport. Likewise, 5aza-C alone reduced the rate of 3MG transport to only 12% of the control values. Simultaneous administration of cytidine in a dose of 50 mg/kg, ip, which is three to four times greater than that of 5-aza-C, prevented the inhibition of 3MG transport and partially prevented the body weight loss caused by 5-aza-C. Cytidine alone had no effect on 3MG transport. DISCUSSION It has been reported tha ara-C, administrated ip to mice in the optimum therapeutic dosage schedule of 15 g/kg/dose, every 3 hr for 3 days, resulted in progressive damage to the crypt epithelial cells of the intestinal mucosa (Leach et al., 1969). Results of the present study demonstrated that ara-C and 5-aza-C, administered in doses comparable to those used clinically, caused generalized impairment of active intestinal transport processes in mice. Both drugs caused profound depression of glucose, amino acid, and Na absorption (associated with decreases in
78
THERESA
S. CHEN
TABLE PROTECTIVE
EFFECTOF
5
2'-DEOXYCYTIDINEAND CYTIDINEON ANTICANCER 3-O-METHYL-D-GLUCOSE (3MG) TRANSPORT“
Treatment
n*
Control 2’-Deoxycytidine Ara-C 2’-Deoxycytidine + Ara-C Cytidine 5-Aza-C Cytidine + 5-Aza-C
I 6 10 9 5 8 6
a Values are x + SE. b Number of animals per group. ’ Change from initial; + indicates d Not significantly different from
3MG transport (wol/g/hr)
weight control.
rate
7.13 6.38 1.06 5.88 6.12 0.88 5.88
+ iz t + f + +
gain,
- indicates
p value
0.68 0.71 0.14 0.63 0.50 0.25 0.50
the transmucosal PD and I,,), as well as a substantial weight loss. This functional impairment was due to a direct toxic effect of the drugs rather than a secondary effect of weight loss, since comparable weight loss caused by fasting actually resulted in significant enhancement of glucose and amino acid absorption. The latter observation is in agreement with the finding of Kershaw et al. ( 1960) that starvation resulted in an enhancement of nutrient absorption in the rat. The anticancer activity of ara-C and 5aza-C is dependent upon phosphorylation in the body with formation of the presumed active cytotoxic species, ara-CTP, and 5aza-CTP (Kessel et al., 1967; Li et al., 1970). The observed lack of effect on intestinal transport function during short-term in vitro exposure suggested that metabolic activation was also involved in gastrointestinal toxicity. This lack of toxicity in vitro was probably the result of insufficient exposure time but could also occur if activation at a remote site was required for the toxic effects on the gastrointestinal tract. However, this is unlikely since the active nucleotides are large ionic compounds which are mainly restricted to an intracellular location (Sadee
NSd co.01 NS NS
weight
DRUGS
INDUCED
Body weight change’ (o/o) +0.98 +I.62 -11.83 +2.03 -0.39 -20.68 -8.15
f + + f -c f +_
0.29 0.87 1.23 0.63 1.66 1.22 2.24
INHIBITIONOF
p value
NS
loss.
and Wong, 1977). Furthermore, preliminary studies in this investigation revealed that treatment for only 1 to 2 days caused no impairment of transport function, suggesting that both the time of exposure and drug concentration are important determinants of toxic effects (Mellett, 1974). Since the time course of morphologic changes (Leach et al., 1969) and functional changes induced by ara-C are similar it is possible that the impairment of intestinal function is a consequence of morphological alterations. The observed gastrointestinal toxicity was largely preventable by pyrimidine nucleosides capable of competitively inhibiting the formation of the active nucleotides of ara-C and 5-aza-C. Lee et al. (1975) demonstrated that in fibrosarcoma and leukemic cells, deoxycytidine and cytidine were potent competitive inhibitors of the phosphorylation of ara-C and 5-aza-C, respectively. It has been shown that simultaneous oral administration of 2’-deoxycytidine in doses two to three times as high as those of ara-C prevented drug-related lethal toxicity in mice yet the antitumor activity of ara-C remained (Buchman et al., 1979). Evans and Hanka (1968) showed that the antileukemic activity of 5-
CYTOTOXIC
DRUGS
ON
azacytidine could be reveresed in vivo by either cytidine or uridine. Similarly, the present study showed that the suppression of glucose absorption and body weight loss caused by ara-C was completely prevented by simultaneous treatment with 2’-deoxycytidine, and the suppression of glucose absorption caused by 5-aza-C was largely prevented by simultaneous administration of cytidine, even though the body weight loss was only partially prevented. Malnutrition is a common clinical finding in cancer patients. While this may be due in part to poorly understood systemic effects of malignant neoplasms, the present investigation demonstrated that anticancer drugs are also capable of causing or aggrevating the condition by depression of intestinal absorptive activity. Furthermore, the protective effects of 2’-deoxycytidine and cytidine on ara-C- and 5-aza-C-induced inhibition of glucose absorption may be beneficial in preventing the related gastrointestinal toxicity by anticancer drugs. ACKNOWLEDGMENTS The author is indebted to Dr. K. C. Huang for his valuable discussion and review of this manuscript and to Dr. P. W. O’Connell of the Upjohn Company for the arabinosyl cytosine. This study was supported in part by American Cancer Society Grant In 1llE and USPHS Grant CA-25252-02.
REFERENCES BUCHMAN, V. M., BELYANCHIKOVA, N. I., MKHEIDZE, D. M., LITOVCHENKO, T. A., LICHINITSER, M. R., BARKHOTKINA, M. F. AND SVET MOLDAVSKY. G. J. (1979). 2’-Deoxycytidine hydrochloride protection of mice against the lethal toxicity of cytosine arabinoside. Cancer Chemother. Pharmacol. 3, 229-234. CAPEL, I. D., JENNER, M., PINNOCK, M. H., ANDWILLIAMS, D. C. (1978). The effect of anti-cancer drugs on the plasma disposition of antipyrine and the biliary excretion of phenolphthalein in the rat. B&hem. Pharmacol. 27, 1413-1416. CAPEL, I. D., PINNOCK, M. H., AND WILLIAMS, D. C. (1979). An in vitro assessment of the effect of cyto-
INTESTINAL
TRANSPORT
79
toxic drugs upon the intestinal absorption of nutrients in rats. Eur. J. Cancer 15, 127-l 31. CHANG, L. R., CHEN, T. S. T., AND HUANG, K. C. (1974). Electrolyte transport across the mouse small intestine. Proc. Sot. Exp. Biol. Med. 145, 1220-1224. CURRAN, P. F., WEBSTER, E. W., AND HOVESPIAN, J. A, (1960). The effect of X-irradiation on sodium and water transport in rat ileum. Radiat. Res. 13, 369-380. ESTENSEN, R. D., AND BASERGA, R. (1966). Puromycin induced necrosis of crypt cells of the small intestine of mouse. J. Cell Biol. 30, 13-22. EVANS, J. S., AND HANKA, L. J. (1968). The in vivo activity of combinations of S-azacytidine and cytidine on leukemia L-1210. Experientia 24, 922-923. KERSHAW, T. G., NEAME, K. D., AND WISEMAN, G. (1960). The effect of semistarvation on absorption by the rat small intestine in vitro and in vivo. J. Physiol. 152, 182-190. KESSEL, D., HALL, T. C., AND WODINSKY, I. (1967). Transport and phosphorylation as factors in the antitumor action of cytosine arabinoside. Science 156, 1240-1241. KOBAYASHI, Y., AND MAUDSLEY, D. V. (1969). Practical aspects of liquid scintillation counting. Methods Biochem. Anal. 17, 55-133. LEACH, W. B., LASTER, W. R., JR., MAYO, J. G., GRISWOLD, D. P., JR., AND SCHABEL, F. M., JR. (1969). Toxicity studies in mice treated with 1-@-D-arabinofuranosylcytosine (ara-C). Cancer Res. 29, 529535. LEE, T., KARON, M., AND MOMPARLER, R. L. (1975). Cellular phosphorylation of l-P-D-arabinofuranosylcytosine and .5-azacytidine with intact fibrosarcoma and leukemic cells. Cancer Res. 35, 2506-2510. LENAZ, L., AND PHILIPS, F. S. (1970). Effects of arabinosylcytosine nucleosides on DNA synthesis in rats. Cancer Res. 30, 1961-1962. LENAZ, L., STERNBERG, S. S., AND PHILIPS, F. S. (1969). Cytotoxic effects of I-B-D-arabinofuranosyl5-fluorocytosine and of l+D-arabinofuranosylcytosine in proliferating tissues in mice. Cancer Res. 29, 1790-1798. LI, L. H., OLIN, E. J., BUSKIRK, H. H., AND REINEKE, L. M. (1970). Cytotoxicity and mode of action of 5azacytidine on Ll210 leukemia. Cancer Res. 30, 2760-2769. LIEBERMAN, M. W., VERBIN, R. S., LANDAY, M., LIANG, H., FARBER, E., LEE, T. N., AND STARR, R. (1970). A probable role for protein synthesis in intestinal epithelial cell damage induced in vivo by cytosine arabinoside, nitrogen mustard, or X-irradiation. Cancer Res. 30, 942-951. MELLETT, L. B. (1974). The constancy of the product of concentration and time. In Antineoplastic and Immunosuppressive Agents (A. C. Sartorelli and D. G.
80
THERESA
Johns, eds.), Part I, pp. 330-340. Springer-Verlag. Berlin. SADEE, W., AND WONG, C. G. (1977). Pharmacokinetics of 5-fluorouracil: Interrelationship with biochemical kinetics in monitoring therapy. Clin. Pharmacokinet. 2, 437-450. SHARMA, R. K., AND NAGCHAUDHURI, J. (1976). A study of amino acid absorption from small intestines of vinblastin treated rats with altered mucosal morphology. Indian J. Med. Rex 64, 1225-1234. SLAVIN, R. E., DIAS, M. A., AND SARAL, R. (1978).
S. CHEN Cytosine arabinoside induced gastrointestinal toxic alterations in sequential chemotherapeutic protocols. Cancer 42, 1747-1759. USSING, H. H., AND ZERAHN, K. (195 1). Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol. &and. 23, 110-127. WILSON, T. H., AND WISEMAN. G. (1954). The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J. Physiol. 123, 116- 125.