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Antitumor agents. I. Effect of 5-fluorouracil and cyclophosphamide on liver microsomes and thymus of rat.

Effects of antitumor agents on rat liver microsomal drug-metabolizing enzyme activities and thymus lymphocytes were studied in male Wistar rats. High doses of 5-fluorouracil (5-FU) and cyclophosphamide (CP) given parenterally for 6 days caused a partial decrease in whole body weight and the microsomal enzyme content such as cytochrome P-450 and cytochrome b5. Aniline p-hydroxylase and aminopyrine N-demethylase activities also decreased in rats dosed for 5 days decreased compared with the control. Both compounds in the high concentrations produced spectral change of "modified type II". However, the magnitude of the spectral changes observed was independent of the the concentration of substrate added. The addition of NADPH to the microsomes-substrate mixture modified the spectral change. Both drugs caused a considerable decrease in thymus weight and the number of thymus lymphocytes, while the alkaline phosphatase activity was enhanced in 5-FU groups, indicating that the agents cause a significant involution of the thymus. Decrease in the total number of the lymphocytes was greater than that in the blood leucocytes.


5-Fluorouracil
and cyclophosphamide (CP) are representative antitumor agents and widely used in cancer therapy. The former, which was first synthesized by Duschinsky et al. (1), inhibits DNA and RNA syntheses (2) as the competitive agent, and the latter, which is non-toxic to cancer cells in vitro, is considered to be first converted, presumably by the microsomal mixed-function oxidases, into 4-hydroxycyclophosphamide which may then break down to yield phosphoramide mustard (3), an active and potent antitumor agent (4). 5-FU has many adverse reactions such as hematopoietic depression, diarrhea, weight loss and hemorrhages in the lungs and intestines (5), while CP with a low toxicity (6) causes a decrease in leucocytes (7).
One of our objectives herein was to establish the effects of the antiturnor agents on the thymus which plays an important role in immuno-defense mechanisms. When antitumor agents are given to cancer patients, the activity of the reticulo-endothelial system generally decreases, consequently the decreased immuno-response may result in a deterioration and development of tumors (8). It is thus of interest to elucidate the influence of antitumor agents on the thymus in terms of drug therapy of a tumor. The thymus in the neonate exerts an essential role in the development of lymphoid tissues and the establishment of immune functions (9,10), and thymectomy of neonatal animals leads to a decrease in immune func tions (11,12). In adult animals, the thymus is related to the establishment and restoration of the immune functions, in particular the production and development of lymphocytes (13).
Thus, the thymus provides resistance against the tuntorigenesis and development of tumor in animals. Although antitt.imor agents do have side effects as described above, damage to the liver is relatively slight in the clinically effective doses (14,15). The second objective was to clarify the mechanism when even a slight lesion was evident in the liver after administration of these antitumor drugs.
Alterations of the drug-metabolizing enzyme activities, thymus alkaline phosphatase activity and the total number of thymus lymphocytes in comparison with blood leucocytes were investigated after high doses of these drugs.  The animals were decapitated 2 hr after the final administration of saline or the drugs at 3, 6 and 10 days. The doses of 5-FU and CP to rats were approx. 3 and 10 times higher than the clinically effective daily doses, respectively.

Materials
Preparation of liver microsomal fractions: The liver microsomal fractions were prepared according to the method of Omura and Sato (16).
Measurements of the total number of blood leucocytes and thynnis lymphocytes: The blood of male Wistar rats was collected from the abdominal aorta into heparin-containing tubes (100 units per ml blood). The whole thymus, which was washed once with saline, was gently homogenized in saline with a glass homogenizer. The total nun-iber of the leucocytes and lymphocytes was counted using a Barker Turk counting chamber.
Protein determination: Protein concentrations of microsomal preparation and the thymus suspension were estimated by the method of Lowry et al. (17).
Assays of enzymes: NADPH-cytochrome c reductase activity was assayed at 20°C by observing the change in absorhance at 550 nm resulting from cytochrome reduction, according to the procedure described by Slater and Sawyer (18). Aniline p-hydroxylase activity was estimated at 37°C according to the method of Ikeda (19). The demethylation of aminopyrine was assayed at 37 3C by the method of Ariyoshi and Takabatake (20), and formaldehyde formed was determined by the method of Nash (21). The concentration of cytochrome P-450 (P-450) and cytochrome b5 (b5) was determined at room temperature with a Shimadzu IMPS-50L spectrophotometer as described by Omura and Sato (16). Alkaline phosphatase activity was determined in rat thymus homogenized in saline. A mixture consisting of 1 ml of p-nitrophenylphosphate (0.015 N1 in the reaction medium) and 1 ml of 0.09 N1 glycine buffer (pH 10.0) was preincubated at 37'C for 10 min and then 0.2 ml of thymus suspension was added. After incubation at 37 C for 20 min, the reaction was termi

Effect of 5-FU and CP on body and liver weights
As shown in Table 1, animals injected with these drugs failed to grow, whereas there was a normal increase in the body weight of control animals, and the weight of animals treated with these drugs decreased at day 6 rather than that on the first day. The cessation of the drug treatment, however, caused a partial increase of the body weight in 5-FU-injected rats at 10 days, while a slight decrease of the weight was observed in CP group which was given the drug in a dose ten times the clinically effective daily dose. The dose was three-fold in the 5-FU group. Relative liver weight (g liver/100 g body weight) was increased slightly after CP treatment but there was no significant increase in the weight of the 5-FU group, as compared with the controls. To clarify the effect of antitumor agents on the activity of the microsomal drug-me tabolizing system, enzyme activities of the microsomes from the groups on the drugs for 3 or 6 days were measured. As shown in Table 2, NADPH-cytochrome c reductase activity was not significantly altered and it would appear that the enzyme is little affected by these drugs or the metabolites. The slight increase in P-450 and b5 levels attributed to the enzyme induction was observed in both groups administered the drugs for 3 days, whereas at 10 days the content of P-450 had significantly decreased, by 2600 in 5-FU group and 48% in CP group, as compared with that of the control group, respectively. The b5 level of CP group was also decreased at 10 days. The decrease in these pigment contents may be due to the decreased syntheses and/or the increased degradation with accumulation of the drug and the toxic metabolites in situ. Aniline p-hydroxylase activity was also decreased 43 %, after administration of CP for 6 days in contrast with the slight decrease in rats dosed 5-FU.
The decreased activity in CP group was restored to 75 of the control value after cessation of administration, at 10 days. The decrease in aminopyrine N-demethylase activity was also shown in CP-treated rats at 10 days, however, there was only a partial change of this activity during administration of these antitumor agents.  A plot of the change of the absorbance at fixed wavelength (420 nm) as a function of 5-FU or CP concentrations was made, and the reciprocals of these data were plotted as il lustrated in Fig. 3. We found that a single daily dose of 5-FU and CP for 6 days produced a remarkable decrease in the thymus weight and the total number of lymphocytes. After termination of administration, the decreased weight and lymphocytes in CP group remained unchanged, in contrast to the partial increase in the 5-FU group at 10 days, as shown in Table 3. How ever, the greater effect of CP on the thymus is no doubt due to the higher close of this drug.
A further increase (50 mg per kg body weight) of a dose of these drugs was found to induce a greater decrease in the thymus weight and the number of the lymphocytes, followed by increase in the lethal rate at 6 days (30% death). Following treatment with 5-FU, the alkaline phosphatase activity in the thymus was greatly increased at 6 days, while the activity in the CP group was only slightly increased at the same day. The increased activity returned to the control level after termination of administration at 10 days. CP given for 3 days induced a decrease in the activity, suggesting that the effect of CP on the thymus may be different from that of 5-FU.

Effect of 5-FU and CP on blood leucocytes
The effect of 5-FU and CP on the blood leucocytes was tested in comparison with that of thymus lymphocytes. The administration of these drugs for 6 days decreased significantly the total number of the leucocytes, although the decreased leucocytes partially recovered after termination of administration, as shown in Table 4. Decrease in the total number of the leucocvtes was much less than that seen in lymphocytes.
DISCUSSION high closes of both ant it unior agents for 6 days were found to cause a significant in volution of the thymus and a decrease in lymphocytes as compared with those of control second day during a period of 10 days) resulted in a cessation of the growth of rats, an almost complete thymolytic reaction and a clearly raised activity of succinate reductase and acid phosphatase of the thymus (23). An elevation of acid phosphatase (24), adenosintriphos phatase and 5-nucleotidase (25) have also been demonstrated biochemically in the rat thymus following X-irradiation. Further, it is known that the activities of (9-glucuronidase (26), catepsin and arylsulfatase (27)  The inductive formation of the drug metabolizing enzymes of rat liver by administration of these drugs was not significant, although a small increase in P-450 and b5 levels was seen at 3 days. Inversely, administration of the drugs, every day on six occasions, caused a significant decrease in aniline p-hydroxylase at 6 days and in P-450 and b5 at 10 days (Table   2). This decrease is ascribed to the inhibition and degradation of the enzymes by the me tabolites, fluoroacetate from 5-FU (28) and phosphoramide mustard from CP (3), which are known to be cytotoxic agents, and to an increased inhibition of protein synthesis due to accumulation of the drugs in situ. It has been reported that toxicity of CP is significantly enhanced by increased accumulation (7). The decreased P-450 content found after 10 days dosing is attributed, at least partially to damage of the electron transport pathway of micro somal mixed function oxidase system, and consequently in a decrease of aniline p-hydroxylase and aminopyrine N-demethylase activities. The fact that no significant effect was found on NADPH-cytochrome c reductase suggests a special property of the enzyme. This assumption is strengthened by previous data that the activity was not inhibited by free radicals and lipid peroxides in carbon tetrachloride intoxication. The observation that these drugs in a higher concentration cause modified type II spectrum as a result of substrate interaction with a hepatic microsomal cytochrome and that the magnitude of the spectral changes is little dependent upon the concentration of substrate added to the microsome suspension suggests that the binding force of the drugs to the microsomes, probably P-450 (22), is fragile (Figs. 2 and 3). The possible explanation for this phenomenon may lie in the highly polar character (CP and 5-FU) or low lipid solubility (5-FU) and may explain the lack of damage to the liver during these drug treatments. The extreme NADPH dependence of the binding of these substrates to microsomes is interesting since reduction of the P-450-substrate complex by NADPH has been suggested as the rate limiting step in the microsomal oxidation of some substrates, such as ethyl niorphine (29) and cyclohexane (30).
Thus these antitumor agents cause significant involution of the thyn-ius and decrease in the lymphocytes.
The administration of the agents for 6 days produced a partial decrease in liver microsomal enzyme activities and such was attributed to the metabolites and the increased accumulation of the drugs. Binding of the drugs to the microsomes was fragile and was strongly affected by addition of NADPH.