Influences of Anticancer Agents on the Metabolism of Δ-Aminolevultnic Acid in Normal and Tumor-Bearing Mice*1,*2

[GANN, 55, 25-40; February, 1964] UDC 615.771.7-092:616-006-092:612.015.3:547.484.3

INFLUENCES OF ANTICANCER AGENTS ON THE METABOLISM OF δ-AMINOLEVULTNIC ACID IN NORMAL AND TUMOR-BEARING MICE*1,*2

Kotobuki HANO and Akira AKASHI (Department of Pharmacology, Faculty of Pharmacy, Osaka University*3)

Synopsis

Previous findings had shown that the effect of anticancer agents in restoring lowered enzyme activities in the liver and especially that of catalase was not always in parallel with their therapeutic effect. To elucidate this, the possible inhibitory effect of these agents on heme metabolism, with special reference to iron, copper,

and δ-aminolevulinic acid metabolism in normal and tumor-bearing mice, was

investigated.

The δ-aminolevulinic acid dehydrase activity in the liver of tumor-bearers was lower than that of normal animals and was found to be very low in tumor cells. Of the anticancer agents tested in vitro, 2, 5-bis(ethyleneimino)-1, 4-benzoquinone and folic acid antagonists were found to inhibit this enzyme activity in normal mice.

On the other hand, carboxamide utilization in which δ-aminolevulinic acid is concerned with the source of a C1-donor occurred at a much higher rate in tumor cells than in the liver of normal and tumor-bearing mice. In the presence of folic acid antagonists, and d-catechin and berberine, carboxamide utilization by tumor cells was markedly inhibited in vitro.

Daily decrease in the serum iron level, blood hemoglobin content, and liver δ- aminolevulinic acid dehydrase activity was observed after tumor transplantation, when the serum copper level increased. Administration of alkylating agents and 8-azaguanine to tumor-bearers restored these metabolic disturbances to normal in parallel with their therapeutic effects. These agents showed no influence on these levels in normal mice. 6-Mercaptopurine, which has a marked anticancer activity

with Ehrlich ascites carcinoma, returned the lowered liver δ-aminolevulinic acid dehydrase activity and elevated serum copper level in tumor-bearing animals to normal. However, the lowered serum iron level and blood hemoglobin content in tumor-bearers were further depressed by treatment with 6-mercaptopurine and this depressive action was also found in normal mice. Treatment with aminopterin, which

*1 This constitutes a part of a series entitled "Pharmacological Studies on the Metabolism of

Cancer Tissues," Part XXXIX. *2 This investigation was supported in part by a Grant-in-Aid for Fundamental Scientific Research from the Ministry of Education, and in part by grant CY-5257 from the National Institutes of Health, U.S.A.. *3 6-5 Toneyama, Toyonaka, Osaka-fu (羽野壽, 明石章)

25 was not effective against Ehrlich ascites carcinoma at the dose level tested, did not restore the altered metabolism of tumor-bearing animals to normal and this agent caused a depression of the blood hemoglobin content in normal mice. The significance of these results in relation to those in the previous report is discussed.

INTRODUCTION

A previous paper8) from our laboratory reported comparative studies on the possible parallelism between the effect of various anticancer agents and their effectiveness in restoring the lowered activities of liver catalase and uricase to normal in mice bearing Ehrlich ascites carcinoma. It was found that the effect on the lowered enzyme activities, especially of catalase, in tumor-bearing mice was not always comparable to the therapeutic effect of these anticancer agents. Both in vivo and in vitro purine and folic acid antagonists inhibited liver catalase in normal mice and did not enhance the enzyme activities. Thus, these agents might inhibit iron and porphyrin metabolism. Both the activity and content of liver catalase and blood hemoglobin, which have heme as the prosthetic group, decrease in tumor-bearing animals.7) Therefore, interest has arisen on the metabolism of δ-aminolevulinic acid (ALA), a specific precursor of heme biosynthesis,3) and on the metabolism of iron, copper, and hemoglobin. It has been established that ALA is converted to porphobilinogen (PBG), a specific precursor of porphyrin, catalyzing ALA dehydrase.5,6) Moreover, ALA is also utilized as the source of the C1-donor for purine biosynthesis from 4-amino-5-imidazole- carboxamide (carboxamide) through the succinate-glycine cycle14,19) (Fig. 1).

Fig. 1. Metabolism of δ-amino-

levulinic acid

26 Studies on the effect of various anticancer agents and some plant components,9) which have been tested for anticancer activity in this laboratory, on ALA metabolism, and changes in the levels of serum iron, copper, and hemoglobin in normal and tumor-bearing mice are reported in this paper.

MATERIALS AND METHODS

The experimental materials were similar to those described in the previous report8) but will be described briefly here for the convenience of readers. The anticancer agents and therapeutic doses used in these experiments were as follows: 1. Alkylating agents: Triethylenethiophosphoramide (Thio-TEPA) 5mg/kg, p- phenylenediphosphoric acid tetraethyleneimide (RC-4) 60mg/kg, 2, 5-bis(ethyleneimino-1, 4-benzoquinone) (DQ). 2. Purine antagonists: 6-Mercaptopurine (6-MP) 80mg/kg, 8-azaguanine (8-AG) 200mg/kg, 2, 6-diaminopurine (2,6-DAP), 6-chloropurine (6-CP). 3. Folic acid antagonists: Aminopterin (A-PGA) 0.5mg/kg, amethopterin (A-M-PGA). For in vivo experiments, male ddO strain mice weighing about 20g were used in three groups of 50 animals. Two groups were inoculated intraperitoneally with 2× 106 Ehrlich ascites carcinoma cells and one of these groups was administered with anticancer agents. Treatment, at the indicated dose levels, was initiated 24 hours after transplantation and continued as a single, daily intraperitoneal injection for 5 days. The third group, which were non-tumor-bearing mice, was also injected with anticancer agents once daily for 5 days. Changes in liver ALA dehydrase activity and levels of serum iron and copper, and blood hemoglobin in each group were estimated daily 18 hours after the injection of anticancer agents for 5 days and then daily for the next 5 days. Five or six mice in each group were sacrificed by decapitation and exsanguinated. The excised liver and pooled blood were used for the following estimations. The in vitro influence of the anticancer agents on the activity of liver ALA dehydrase in mice and on the carboxamide utilization of Ehrlich ascites carcinoma cells were examined by adding these agents to reaction mixtures at a final concentration of 2×10-3 to 2×10-4M.

In experiments on the metabolic activity of ALA in normal and tumor-bearing mice, rats bearing ascites hepatoma AH-130 were also used. In this case, the ascitic fluid was harvested from rats inoculated with ascites hepatoma cells 7-8 days previously and 0.5ml of the ascitic fluid was transplanted into male albino rats of random breed weighing 200-250g. 1. Assay of ALA Dehydrase Activity A liver homogenate was prepared by mixing the liver with 9 times its wet weight

27 of ice-cold 0.15M KCl and blending the mixture in a Potter-Elvehjem glass homoge- nizer. Ehrlich ascites carcinoma cells and ascites hepatoma cells were harvested 7- 8 days after intraperitoneal transplantation. The cells were washed with ice-cold physiological saline and the red cells were separated by low-speed centrifugation. The cells obtained were resuspended in 4 volumes of 0.15M KCl.

Liver homogenates and cell suspensions were incubated with 8×10-3M ALA and

0.15M KCl in a total volume of 5.0ml for 1 hour at 37.5° under anaerobic conditions.

Then an equal volume of 10% trichloroacetic acid was added to stop the reaction. The mixture was centrifuged at 2,000g. The porphobilinogen in the supernatant was determined by the method of Gibson et al.5) 2. Assay of Carboxamide Utilization Enzymes were prepared as in 1 and the enzyme preparations were incubated

with 5×10-3M carboxamide, 5×10-3M ALA, 1×10-2M ATP, 1×10-2M sodium succinate,

1×10-2M MgCl2, and 0.15M KCl in a total volume of 3.0ml for 1 hour at 37.5° under aerobic conditions. The reaction was stopped by the addition of an equal volume of 20% trichloroacetic acid and the residual carboxamide in the supernatant was determined by the Bratton-Marshall diazotization reaction, as modified by Ravel et al.16) 3. Assay of Blood Hemoglobin, Serum Iron, and Copper The blood hemoglobin concentration was measured with Sahli's hemometer. After assay of the hemoglobin content, the blood from each group of mice was pooled and

allowed to stand overnight at 5°. Then it was centrifuged at 2,000g and the serum obtained was used for iron and copper determination. The method of Schade et al.17) was slightly modified for the determination of serum iron. Aliquots of 1.0ml of serum were pipetted into 2 test tubes, containing 2.0ml of Reagent A*4 or B,*5 respectively. Then 2.0ml of distilled water was added and the tubes were placed in

a water bath of 45° for 20 minutes. The color developed was measured in a Beck-

man DU spectrophotometerat 552mμ. Serum copper was measured by the method of Eden and Green4) in which 0.1% sodium diethyldithiocarbamate was added to 1.0ml of the serum sample and the color developed in a total volume of 4.0ml was determined spectrophotometrically at 440mμ.

RESULTS

I. Effect of Anticancer Agents on the Metabolism of ALA in vitro (1) Effect on Liver ALA Dehydrase Activity of Normal Mice: Effect of various anticancer agents on the enzyme activity in vitro is summarized in Fig. 2. DQ and *4 A mixture of 4 parts of phosphate buffer-ascorbic acid reagent and 6 parts of water, for the control sample. *5 A mixture of 4 parts of phosphate buffer-ascorbic acid reagent and 2 parts of terpyridine reagent, for the test sample. 28 folic acid antagonists reduced the enzyme activity on an average of approximately 40 and 20%, respectively, and other agents tested had no effect.

Fig. 2. Effect of anticancer agents on the activity of liver ALA dehydrase of normal mice in vitro

(2) Effect on Carboxamide Utilization by Tumor Cells: Carboxamide utilization was not influenced to a singnificant degree in the presence of alkylating agents and purine antagonists, but folic acid antagonists caused 70-80% inhibition, as shown in Fig. 3. Of the plant components tested, including flavonoids, lycoris alkaloids, and others, only d-catechin (flavonoid) and berberine inhibited carboxamide utilization (Fig. 4).

Fig. 3. Effect of anticancer agents on the utilization of carboxamide by Ehr- lich ascites tumor cells in vitro

29 Fig. 4. Effect of some plant components on the utilization of carboxamide by Ehrlich ascites tumor cells in vitro

II. Effect of Anticancer Agents on the Metabolism of ALA, Serum Iron and Cop- per Levels, and Hemoglobin Content in Normal and Tumor-bearing Animals (1) ALA Dehydrase Activity in Liver and Tumor Cells: As shown in Figs. 5 and 6, ALA dehydrase, which functions in the conversion of ALA to PBG, was less active

Fig. 5. Relative activity of ALA dehydrase of liver and tumor cells in mice

30 Fig. 6. Relative activity of ALA dehydrase of liver and tumor cells in rats

in the liver of tumor-bearing animals than in normal animals. Its activity in Ehrlich ascites carcinoma cells and ascites hepatoma cells was very low. (2) Utilization of Carboxamide by Liver and Tumor Cells: Fig. 7 shows that liver homogenates of normal and tumor-bearing mice utilized carboxamide at the rate of about 3.7 and 4.2mμmole/mg dry weight/hour and high carboxamide utilization, averaging 5.0mμmoles, occurred in tumor cells. Slightly higher values were obtained with rat ascites hepatoma cells (Fig. 8).

Fig. 7. Utilization of carboxamide by liver and tumor cells in mice

31 Fig. 8. Utilization of carboxamide by liver and tumor cells in rats

(3) Liver ALA Dehydrase Activity, Serum Iron and Copper Levels, and the Blood Hemoglobin Content of Tumor-bearing Mice: A gradual daily depression in the liver

Fig. 9. Liver ALA dehydrase activity, serum iron and copper levels, and blood hemoglobin content in tumor-bearing mice Each point is the mean of the values of 5 or 6 mice.

32 ALA dehydrase activity, and the serum iron and blood hemoglobin contents was observed after tumor transplantation and values reached about 60, 35, and 70%, respectively, of normal on the 10th day. On the other hand, the serum copper level increased considerably (Fig. 9).

□---□ tumor-bearers △-△ tumor-bearers □----□ tumor-bearers+agent △----△ tumor-bearers+agent

■----■ normal mice+agent ▲----▲ normal mice+agent

○-○ tumor-bearers ●-● tumor-bearers

Fe {〇----○ tumor-bearers+agent Cu ●----● tumor-bearers+agent ○----○ normal mice+agent {●----● normal mice+agent

Fig. 10. Effect of Thio-TEPA on the liver ALA dehydrase activity, serum iron and copper levels, and blood hemoglobin content Each point is the mean of the values of 5 or 6 mice. These symbols apply to all subsequent graphs.

33 (4) Influence of Anticancer Agents on the Liver ALA Dehydrase Activity and the Serum Iron and Copper and Blood Hemoglobin Contents: a) Alkylating Agents: Changes in the ALA dehydrase activity serum iron, and copper, and in hemoglobin content of normal and tumor-bearing mice following administration of these agents are shown in Figs. 10 and 11. The lowered ALA dehydrase activity, iron and hemoglobin levels, and the increased copper level were restored almost to normal. The effect of these agents was parallel to their therapeutic effect. These agents had no effect on normal mice.

Fig. 11. Effect of RC-4 on the liver ALA dehydrase activity, serum iron and copper levels, and blood hemoglobin content b) Purine Antagonists: Of the purine antagonists tested, 6-mercaptopurine restored the lowered activity of liver ALA dehydrase and the increased serum copper level of tumor-bearers to normal, this effect being parallel to its therapeutic effect. How- ever, the serum iron level and hemoglobin content, which decreased after tumor

34 inoculation, decreased further during administration of 6-mercaptopurine and there was no subsequent return to the normal level (Fig. 12). Fig. 12 also shows that 6-mercaptopurine caused depression of the serum iron and blood hemoglobin contents in normal mice but had no influence on the liver ALA dehydrase activity or serum copper level.

Fig. 12. Effect of 6-mercaptopurine on the liver ALA dehydrase activity, serum iron and copper levels, and blood hemoglobin content

After injection of 8-azaguanine, which has a weaker therapeutic effect than 6- mercaptopurine, these metabolic disturbances of tumor-bearing mice were restored to normal. This agent did not influence the levels in normal mice, indicating a resemblance in its mode of action to that of alkylating agents (Fig. 13).

35 Fig. 13. Effect of 8-azaguanine on the liver ALA dehydrase activity, serum iron and copper levels, and blood hemoglobin content c) Folic Acid Antagonist: As shown in Fig. 14, the altered metabolism of tumor- bearers was unaffected by aminopterin, which is relatively ineffective against Ehrlich ascites carcinoma. As in the case of 6-mercaptopurine, aminopterin caused a decrease in the blood hemoglobin content in normal mice.

DISCUSSION

In agreement with the observations of others,12,20) our results showed that ALA dehydrase which is involved in the first step of ALA metabolism was less active in the liver of tumor-bearing animals and tumor cells than in normal animals. These results may help to elucidate the well-known fact that the levels of both liver cata-

36 Fig. 14. Effect of aminopterin on the liver ALA dehydrase activity, serum iron and copper levels, and blood hemoglobin content lase and blood hemoglobin, which have heme as the prosthetic group, decrease in tumor-bearing animals. The previous report showed that the lowered liver catalase activity in tumor- bearers was further depressed by 6-mercaptopurine and 8-azaguanine, in spite of their considerable therapeutic effect, and that the restoration of the enzyme activity to normal was delayed.8) Therefore, these agents might inhibit the synthesis of porphyrin, the prosthetic group of catalase. However, it was found that liver ALA dehydrase activity was not inhibited by these agents in vitro and, therefore, 6- mercaptopurine and 8-azaguanine must inhibit the catalase in a different way. When purine is synthesized from carboxamide and ALA, the other pathway of ALA metabolism in mammals, the rate of carboxamide utilization is considerably high in tumor cells, and had a slight tendency to be elevated in the liver of tumor- bearers. Conzelman et al.2) and Payne et al.15) also reported that the rate of incorpo-

37 ration of 14C-carboxamide into nucleic acid was abnormally high in the liver of tumor-bearing animals. The rate of the two metabolic pathways of ALA in tumor cells is reciprocal. This suggests that the metabolic pattern in tumor cells tends in the direction for rapid

proliferation. This reciprocal relation in the rate of ALA metabolism was also observed in the liver of tumor-bearers, indicating a resemblance in the metabolic

patterns of tumor cells and the cells of the liver of the host. These data support "Greenstein's second law."13)

Because of the vigorous carboxamide utilization in tumor cells, the in vitro effect of known anticancer agents and some plant components was investigated. The reaction was blocked by folic acid antagonists, berberine, and d-catechin, the two latter being the most effective of the various plant components screened for anticancer activity in our laboratory.9) It is of interest to compare these results with that of our earlier findings on the inhibitory effect of berberine on nucleic acid synthesis in tumor cells.10) Therefore, it might be possible to obtain an anticancer agent from among the substances which inhibit this reaction. To study the relationship between the delayed effect of purine antagonists tested on the restoration of the lowered liver catalase activity of tumor-bearers8) and the possible inhibition of porphyrin metabolism by these agents, influences of various anticancer agents on the serum levels of iron and copper, which are involved in the biosynthesis of heme, as well as ALA dehydrase, and on the blood hemoglobin content were determined in normal and tumor-bearing mice. A daily decrease in liver ALA dehydrase activity, serum iron, and blood hemo-

globin was observed in mice bearing Ehrlich ascites carcinoma while the serum copper level increased gradually as the tumor developed. The alkylating agents tested, which restored the lowered liver catalase level in tumor-bearers to normal,8) did not affect the metabolism of normal mice and they restored the altered metabolism of tumor- bearers to normal. These effects were in parallel with their therapeutic effect. On the other hand, 6-mercaptopurine, which delayed the return of the lowered liver catalase activity to normal in tumor-bearers,8) caused a further decrease in the serum iron and blood hemoglobin levels of tumor-bearers and these values did not return to normal even by the end of the experiment on the 10th day. The decreased liver ALA dehydrase and elevated serum copper level in tumor-bearers were restored to normal by 6-mercaptopurine as with alkylating agents. The effect of administration of this agent was the same in normal and tumor-bearers; serum iron and blood hemoglobin were depressed during the period of the treatment. The decrease of iron level in the serum of 6-mercaptopurine-treated mice at the same time as the low blood hemoglobin level suggests either an impairment in iron metabolism or a deficiency in the prosthetic group of heme-protein. However, since

38 6-mercaptopurine does not decrease liver ALA dehydrase activity or the serum copper level, this agent may inhibit iron metabolism in both normal and tumor- bearing mice. In contrast, 8-azaguanine, which also delayed the restoration of the lowered catalase activity in tumor-bearers to normal8) like 6-mercaptopurine, restored the altered metabolism of tumor-bearers to normal, indicating that this agent has no influence on iron and porphyrin metabolism. Therefore, the catalase depression by this agent may not be due to inhibition of iron and porphyrin metabolism but to an interference with biosynthesis of the enzyme protein. A similar result was obtained with 3-amino- 1,2,4-triazole11) which inhibited liver catalase activity both in vitro and in vivo. The results obtained with 6-mercaptopurine and 8-azaguanine suggest that the two agents differ in their mode of inhibition of liver catalase. Treatment with aminopterin, which is not effective against Ehrlich ascites carcinoma at the dose level tested, resulted in the failure of these metabolic disturbances in tumor-bearers to return to normal and the blood hemoglobin level of normal mice was depressed. Thus, the effectiveness of the anticancer agents used in these experiments in restoring the lowered liver ALA dehydrase activity to normal was in parallel with their therapeutic effect. As similar results could probably be obtained with other anticancer agents, this enzyme reaction could probably be used as a supplementary screening test for anticancer substances with regard to the difference in response of tumor cells and host tissues to anticancer agents. A milky turbidity which seemed to be caused by hyperlipemia1) was noted in the serum of mice bearing Ehrlich ascites carcinoma. This phenomenon appeared 6-7 days after the tumor was transplanted and decreased when animals were treated with effective anticancer agents. The influence of anticancer agents on lipid metabolism of tumor-bearers should be studied. Anticancer agents, including alkylating agents, purine antagonists, and folic acid antagonist, had a different effect on the altered metabolism, especially of iron, in the tissues of tumor-bearing mice. Therefore, partial restoration to normal of these impaired iron metabolism in the host tissues resulting from the tumor by non- carcinostatic substances such as iron compounds, might potentiate the treatment with anticancer agents. From this point of view, studies on the potentiation of anticancer agents by various agents are being conducted in our laboratory and results will be published in the near future. (Received September 7, 1963)

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