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Effect of antimetabolites on the growth of Entamoeba histolytica
Experimental
648
EFFECT
OF ANTIMETABOLITES OF ENTAMOEBA IV.
Cell Research 25, 618-6C3 (1961)
ON THE
GROWTH
HISTOLYTICA
FOLIC ACID ANALOGUES M. NAKAMURA
Department
of Microbiology,
Montana
State University,
Missoula,
Montana,
U.S.A.
Received May 20, 1961
FORmany years attempts have been made to elucidate the metabolic pathways of Entamoeba histolytica in order that this protozoan might be grown in pure, bacteria-free cultures and also in order that a more rational chemotherapy might be established. Although several workers [4, 121 have established cultures of E. histolytica for limited periods of growth in environments containing few or no bacteria, little was known about the specific growth factor requirements of this parasite. Studies in adenosinetriphosphate and ribose-5-phosphate fortified, bacteria-inhibited cultures [5] of E. histolytica established the existence of a number of growth factors which were highly stimulatory to the amoebae [S]. The importance of these growth factors was further indicated by a series of studies on the effects of antimetabolites on the growth and multiplication of the amoebae [B, 7, lo]. Since folic acid and citrovorum factor stimulated growth of amoebae [8], it seemed logical to study the activity of folic acid analogues which have become available during the past several years and have been found to inhibit growth of other organisms. MATERIALS
AND
METHODS
The following method was used to evaluate the antimetabolite agents as growth inhibitors. A basal medium consisting of an egg slant overlaid with horse serumRinger solution (4.0 ml per tube), rice powder (approximately lo-15 mg), adenosinetriphosphate (0.1 mg per tube), ribose-5-phosphate (0.2 mg per tube), sodium thioglycolate (5.0 mg per tube), penicillin G (1000 units per tube), and streptomycin (1000 units per tube) was prepared and dispensed in screw-cap tubes (thus eliminating the troublesome vaspar seal [ll]). This medium has been shown to permit excellent growth of amoeba in the absence of detectable viable bacteria. The experimental tubes contained the antimetabolites being assayed; control tubes consisted of basal media without the antimetabolites. All tubes were inoculated with amoebae obtained from 48-72 hr stock cultures growing in association with a mixed bacterial flora. Experimental
Cell Research 25
Anfimefabolifes on the growfh of Enfamoeba hisfolyfica
649
The amoebae were washed three times in Ringer solution by centrifugation before being used as inocula. The tubes were incubated at 37°C for 5-6 days; then most of the overlay fluid was removed with a capillary pipette leaving approximately 1 ml which was thoroughly mixed with the cells without bubbling and a sample of approximately 0.1 ml was placed on a clean slide for counting. A cover slide was placed upon the sample and 10 fields were counted; two samples were taken from each tube, The counting was performed by recording the number of amoebae per low power field. Two strains, HUS-100 and HUS-105, of E. histolytica were employed in these experiments; Dr. Chia-tung Pan, Harvard School of Public Health, Boston, Massachusetts, kindly supplied these cultures. The inhibitory activity of these compounds was recorded as per cent inhibition of amoebic populations at the end of 5-6 days as compared to the controls. Concentrations of the agents necessary for 50 and 100 per cent inhibition were recorded. Reversal studies were performed with folic acid, leucovorin, adenine, guanine, thymine, thymidine, adenosine, guanosine, guanylic acid, thymidylic acid, adenylic acid, methionine, and crystalline vitamin B,,. The following metabolite antagonists were studied: 4-amino-lo-methylpteroylglutamic acid (AMPGA), 9-methylpteroylglutamic acid (MPGA), 9,10-dimethylpteroylglutamic acid (DMPGA), dichloroaminopterin, and 4-amino-pteroylaspartic acid (APAA), obtained from Dr. James M. Smith, Jr., Organic Chemical Research Section, Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York; 2-amino-4-hydroxy-6(1,2,3-trihydroxypropyl-pteridine) (Biopterin), obtained from Dr. H. P. Broquist, Lederle Laboratories Division; aminopterin and sodium pteroyltriglutamate (Teropterin), obtained from Dr. J. M. Ruegsegger, Clinical Research Section, Lederle Laboratories Division; oxyfolic acid, 2-carbethoxy-4,6-diamino-S-triazine (CDT) and 2-carbethoxy-4-phenylamino-6-amino-S-triazine (CPAT), obtained from Dr. Gustav J. Martin, Research Laboratories, National Drug Company, Philadelphia, Pennsylvania. Several of the analogs tested were amoebae-stimulatory; these agents were retested in media not containing the growth factors adenosinetriphosphate and ribose-5phosphate and the activities were recorded in terms of numbers of amoebae per low power field and compared to the amoeba populations in the basal media (without growth factors). RESULTS
A number of folic acid analogues inhibited growth of E. hisfolytica (Table I). The concentrations required for inhibition varied considerably from compound to compound, i.e., 1.0 mg/ml to 0.01 mg/ml for 100 per cent inhibition. Aminopterin, oxyfolic acid, dichloroaminopterin, and AMPGA possessed highest amoebicidal activity (0.001 mg/ml for 50 per cent inhibition and 0.01 mg/ml for 100 per cent inhibition). A tenfold increase in concentration was required, in the cases of these compounds, to produce 100 per cent inhibition as compared to the 50 per cent inhibition level. Benzimidazole, 2,4-diamino-6(N4 sulfanilamido-S-triazine), and 2-carbethoxy-4,6-diaminoExperimental
Cell Research 25
M. Nakamura
650
S-triazine were the least active against the amoebae. A concentration of 1.0 mg/ml (or 100 times the concentration to produce similar effects due to aminopterin) was required to inhibit the amoebae. It is possible that these agents were less effective due to lack of penetration into the cells or possibly by being tied up by cellular constituents in such a way that the agents were not available for the inhibitory effects. The inhibitory effects of many of these compounds were antagonized by various metabolites (Table II). The activity of aminopterin and dichloroTABLE
I. Inhibition
of E. histolytica
in vitro
by some folic acid analogues. Concentrations 50 y0 Inhibition, w/ml
Analogue
0.001 0.001 0.001 0.001 0.001 0.001 0.1 0.1 0.03 0.1
Aminopterin Oxyfolic acid 9,10-dimethylpteroylglutamic acid 4-amino-lo-methylpteroylglutamic acid Dichloroaminopterin 9-methyl-pteroylglutamic acid 2,4-diamino-6(N* sulfanilamido-S-triazine) 2-carbethoxy-4,6-diamino-S-triazine 2-carbethoxy-4-phenylamino-6-amino-S-triazine Benzimidazole
TABLE
II. Reversal of E. histolytica
inhibition
0.01 0.01 0.05 0.01 0.01 0.10 1 .o 1.0 0.1 1.0
by folic acid analogues.
Compounds
Analogue
producing 100 % Inhibition, w/ml
able to reverse inhibition
Aminopterin Dichloroaminopterin
Thymidine, guanylic acid, folic acid, leucovorin, adenosine, guanosine, thymidylic acid
Oxyfolic
Folic acid, leucovorin
acid
Benzimidazole
Adenine,
9,10-dimethylpteroylglutamic acid 4-amino-lo-methylpteroylglutamic acid 9-methylpteroylglutamic acid
Folic acid, leucovorin
2,4-diamino-6(N4 sulfanilamido-S-triazine) 2-carbethoxy-4,6-diamino-S-triazine 2-carbethoxy-4-phenylamino-6-amino-S-triazine
Experimental
Cell Research 25
guanine
I None of the compounds hibition
tested reversed
in-
Antimetabolites on the growth of Entamoeba histolytica
651
aminopterin was reversed by thymidine, guanylic acid, folic acid, leucovorin, adenosine, guanosine, and thymidylic acid, but not by methionine, adenine, guanine, thymine, or vitamin B,,. This is highly suggestive that the amoebae lack the enzyme system(s) for the conversion of purine and pyrimidine bases to their corresponding nucleosides or nucleotides. It is also possible that the stage of inhibition produced by aminopterin and dichloroaminopterin is at TABLE
III.
Stimulation
of E. histolytica
Compound Cont. of compound (mg/ml) . . . Biopterin Teropterin
0 0.1 0.1
growth
by Biopterin
and Teropterin.
Amoebae per low power field 0.01 0.1 0.5 1.0
2.0
10.7 21.0
72.2 82.7
39.0 55.9
50.3 84.4
77.4 80.0
a point further along in the biosynthesis of nucleic acids than at the stage of ribosidation of the bases. Oxyfolic acid, AMPGA, DMPGA, and MPGA inhibition was reversed by folic acid and leucovorin but not by the other metabolites tested. One explanation could be that the inhibitory agents blocked more than one step thus requiring the addition of the preformed final Adenine and guanine growth factor, namely, folic acid and leucovorin. neutralized the toxic activity of benzimidazole. It was not neutralized by the other metabolites tested. Possibly the inhibitory activity of benzimidazole is due to other than blockage of folic acid synthesis or activity. None of the reversal compounds tested reduced the toxicity towards the amoebae of CDT, CPAT, and 2,4-diamino-G(N4 sulfanilamido-S-triazine). Biopterin and Teropterin were stimulatory for amoebic growth at concentrations of 0.1 to 1.0 mg/ml. The growth response of the amoebae to these two agents is recorded in Table III. Addition of adenosinetriphosphate and ribose-5-phosphate to the media containing Biopterin and Teropterin did not significantly increase amoebic numbers in the cultures. APAA was neither stimulatory nor inhibitory to E. histolytica. DISCUSSION
In view of the fact that folic acid and leucovorin are growth factors for E. histolytica, it was not surprising that aminopterin, dichloroaminopterin, oxyfolic acid, AMPGA, MPGA, and DMPGA were inhibitory to this organism. Dewey, Kidder, and Parks [2] found that Tetrahymena could be inhibited Experimental
Cell Research 25
652
M. Nakamura
by 4-amino-9-methylpteroylglutamic acid, 4-amino-lo-methylpteroylglutamic acid, and 4-amino-O,lO-dimethylpteroxylglutamic, ac.id. Folic acid released the Tetrahymena from inhibition by these compounds. Furthermore, aminopterin also reversed the activity of the antifolic which could not be demonstrated in our studies. Franklin and others [3] reported that aminopterin coli in spite of the fact that the organism does not need inhibited Escherichia folic acid for growth. Aminopterin inhibition, in this case, was reversed by thymidine, but not by folic acid, vitamin Blz, thymine, guanine, hypoxanthine, adenosine, adenylic acid, or cytidylic acid. It is possible that in microorganisms not requiring folic acid, the mechanism of action of aminopterin is at another locus since these workers found that Lactobacillus leichmanni 313 which does require folic acid can be protected from aminopterin toxicity by folic acid and thymidine. Since both aminopterin and dichloroaminopterin inhibition was overcome by thymidine, guanylic acid, thymidylic acid, adenosine, and guanosine, a possible interpretation of the data is that these agents block incorporation of both purines and pyrimidines in E. histolytica. Another strong possibility is that aminopterin and dichloroaminopterin block the activity of leucovorin, which indirectly interferes with either necessary for formate transport, utilization or interconversion of purines and pyrimidines. In some strains Nakamura [7] has found that nucleotides and nucleosides of E. histolytica, are necessary growth factors, whereas, in other strains, synthesis of these intermediates can originate de nova. The mechanism of inhibition of CDT, CPAT, and 2,4-diamino-B(N4 sulfanilamido-S-triazine) is not clear from these experiments. None of the metabolites studies reversed this inhibition. It seems likely that these chemicals are directly cytotoxic and are not involved in metabolic antagonisms. However, further experiments are required before definite conclusions can be drawn regarding these compounds. The observation that Biopterin and Teropterin were amoeba-stimulatory was not completely surprising since Broquist and Albrecht [ 1 ] showed that fasciculata, a trypanosomid Biopterin was a growth requirement for Crithidia flagellate. It is possible that Biopterin can replace folic acid as does aminopterin for Tetrahymena [2, 131. The fact that benzimidazole inhibition was not reversed by folic acid or leucovorin suggests that benzimidazole does not directly interfere with the action of folic acid but perhaps prevents purine ring closure or utilization of the purine ring. The role of adenine and guanine in releasing the amoebae from benzimidazole inhibition strengthens this concept. Experimental Cell Research 25
Antimetabolites on the growth of Entamoeba histolytica SUMMARY
Aminopterin, dichloroaminopterin, oxyfolic acid, 9,10-dimethylpteroylglutamic acid, 4-amino-lo-methylpteroylglutamic acid, and 9-methylpteroylglutamic acid inhibited Entamoeba histolytica in uitro. The inhibition was reversed by folic acid and leucovorin. 2,4-diamino-B(N4 sulfanilamido-Striazine), 2-carbethoxy-4,6-diamino-S-triazine, and 2-carbethoxy-4-phenylamino-6-amino-S-triazine were also amoebicidal but reversal of this toxicity was not possible with numerous metabolites studied. 2-amino-4-hydroxy-6(1,2,3-trihydroxypropyl-pteridine) and sodium pteroyltriglutamate stimulated growth of amoebae. Possible mechanisms of action of these chemical agents are discussed. REFERENCES 1. BROQUIST, H. P. and ALBRECHT, A. M., Proc. Sot. Exptl. Biol. Med. 89, 178 (1955). 2. DEWEY. V. C.. KIDDER, G. W. and PARKS, R. E., JR., ibid. 78. 91 (1951). 3. FRANK&N, A. i., STOK~TAD, E. L. R., HOF~MANN; C. e., BELT,‘M. aid JOKES, T. H., J. Chem. sot. 71, 3549 (1949). 4. JACOBS, L., Am. J. Hyg. 46, 172 (1947). 5. NAKAMURA, M., Proc. Sot. EzptZ. Biol. Med. 89, 680 (1955).
6. __ 7. -
ibid. 95, 524 (1957). Biol. Bull. 112, 377 (1957). 8. NAKAMURA, M. and BAKER, E. E., Am. J. Hyg. 64, 12 (1956). 9. Proc. Sot. Exptt. Biol. Med. 92, 723 (1956). 10. NAKAMURA, M. and JONSSON, S., Arch. Biochem. Biophys. 66, 183 (1957). 11. REEVES, R. E., MELENEY, H. E. and FRYE, W. W., Am. J. Hyg. 66, 56 (1957). 12. SHAFFER, J. G. and FRYE, W. W., ibid. 47, 214 (1948). 13. TITTLER, I. A. and BELSKY, M. M., Science 114, 493 (1951).
Experimental
Cell Research 25
648
EFFECT
OF ANTIMETABOLITES OF ENTAMOEBA IV.
Cell Research 25, 618-6C3 (1961)
ON THE
GROWTH
HISTOLYTICA
FOLIC ACID ANALOGUES M. NAKAMURA
Department
of Microbiology,
Montana
State University,
Missoula,
Montana,
U.S.A.
Received May 20, 1961
FORmany years attempts have been made to elucidate the metabolic pathways of Entamoeba histolytica in order that this protozoan might be grown in pure, bacteria-free cultures and also in order that a more rational chemotherapy might be established. Although several workers [4, 121 have established cultures of E. histolytica for limited periods of growth in environments containing few or no bacteria, little was known about the specific growth factor requirements of this parasite. Studies in adenosinetriphosphate and ribose-5-phosphate fortified, bacteria-inhibited cultures [5] of E. histolytica established the existence of a number of growth factors which were highly stimulatory to the amoebae [S]. The importance of these growth factors was further indicated by a series of studies on the effects of antimetabolites on the growth and multiplication of the amoebae [B, 7, lo]. Since folic acid and citrovorum factor stimulated growth of amoebae [8], it seemed logical to study the activity of folic acid analogues which have become available during the past several years and have been found to inhibit growth of other organisms. MATERIALS
AND
METHODS
The following method was used to evaluate the antimetabolite agents as growth inhibitors. A basal medium consisting of an egg slant overlaid with horse serumRinger solution (4.0 ml per tube), rice powder (approximately lo-15 mg), adenosinetriphosphate (0.1 mg per tube), ribose-5-phosphate (0.2 mg per tube), sodium thioglycolate (5.0 mg per tube), penicillin G (1000 units per tube), and streptomycin (1000 units per tube) was prepared and dispensed in screw-cap tubes (thus eliminating the troublesome vaspar seal [ll]). This medium has been shown to permit excellent growth of amoeba in the absence of detectable viable bacteria. The experimental tubes contained the antimetabolites being assayed; control tubes consisted of basal media without the antimetabolites. All tubes were inoculated with amoebae obtained from 48-72 hr stock cultures growing in association with a mixed bacterial flora. Experimental
Cell Research 25
Anfimefabolifes on the growfh of Enfamoeba hisfolyfica
649
The amoebae were washed three times in Ringer solution by centrifugation before being used as inocula. The tubes were incubated at 37°C for 5-6 days; then most of the overlay fluid was removed with a capillary pipette leaving approximately 1 ml which was thoroughly mixed with the cells without bubbling and a sample of approximately 0.1 ml was placed on a clean slide for counting. A cover slide was placed upon the sample and 10 fields were counted; two samples were taken from each tube, The counting was performed by recording the number of amoebae per low power field. Two strains, HUS-100 and HUS-105, of E. histolytica were employed in these experiments; Dr. Chia-tung Pan, Harvard School of Public Health, Boston, Massachusetts, kindly supplied these cultures. The inhibitory activity of these compounds was recorded as per cent inhibition of amoebic populations at the end of 5-6 days as compared to the controls. Concentrations of the agents necessary for 50 and 100 per cent inhibition were recorded. Reversal studies were performed with folic acid, leucovorin, adenine, guanine, thymine, thymidine, adenosine, guanosine, guanylic acid, thymidylic acid, adenylic acid, methionine, and crystalline vitamin B,,. The following metabolite antagonists were studied: 4-amino-lo-methylpteroylglutamic acid (AMPGA), 9-methylpteroylglutamic acid (MPGA), 9,10-dimethylpteroylglutamic acid (DMPGA), dichloroaminopterin, and 4-amino-pteroylaspartic acid (APAA), obtained from Dr. James M. Smith, Jr., Organic Chemical Research Section, Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York; 2-amino-4-hydroxy-6(1,2,3-trihydroxypropyl-pteridine) (Biopterin), obtained from Dr. H. P. Broquist, Lederle Laboratories Division; aminopterin and sodium pteroyltriglutamate (Teropterin), obtained from Dr. J. M. Ruegsegger, Clinical Research Section, Lederle Laboratories Division; oxyfolic acid, 2-carbethoxy-4,6-diamino-S-triazine (CDT) and 2-carbethoxy-4-phenylamino-6-amino-S-triazine (CPAT), obtained from Dr. Gustav J. Martin, Research Laboratories, National Drug Company, Philadelphia, Pennsylvania. Several of the analogs tested were amoebae-stimulatory; these agents were retested in media not containing the growth factors adenosinetriphosphate and ribose-5phosphate and the activities were recorded in terms of numbers of amoebae per low power field and compared to the amoeba populations in the basal media (without growth factors). RESULTS
A number of folic acid analogues inhibited growth of E. hisfolytica (Table I). The concentrations required for inhibition varied considerably from compound to compound, i.e., 1.0 mg/ml to 0.01 mg/ml for 100 per cent inhibition. Aminopterin, oxyfolic acid, dichloroaminopterin, and AMPGA possessed highest amoebicidal activity (0.001 mg/ml for 50 per cent inhibition and 0.01 mg/ml for 100 per cent inhibition). A tenfold increase in concentration was required, in the cases of these compounds, to produce 100 per cent inhibition as compared to the 50 per cent inhibition level. Benzimidazole, 2,4-diamino-6(N4 sulfanilamido-S-triazine), and 2-carbethoxy-4,6-diaminoExperimental
Cell Research 25
M. Nakamura
650
S-triazine were the least active against the amoebae. A concentration of 1.0 mg/ml (or 100 times the concentration to produce similar effects due to aminopterin) was required to inhibit the amoebae. It is possible that these agents were less effective due to lack of penetration into the cells or possibly by being tied up by cellular constituents in such a way that the agents were not available for the inhibitory effects. The inhibitory effects of many of these compounds were antagonized by various metabolites (Table II). The activity of aminopterin and dichloroTABLE
I. Inhibition
of E. histolytica
in vitro
by some folic acid analogues. Concentrations 50 y0 Inhibition, w/ml
Analogue
0.001 0.001 0.001 0.001 0.001 0.001 0.1 0.1 0.03 0.1
Aminopterin Oxyfolic acid 9,10-dimethylpteroylglutamic acid 4-amino-lo-methylpteroylglutamic acid Dichloroaminopterin 9-methyl-pteroylglutamic acid 2,4-diamino-6(N* sulfanilamido-S-triazine) 2-carbethoxy-4,6-diamino-S-triazine 2-carbethoxy-4-phenylamino-6-amino-S-triazine Benzimidazole
TABLE
II. Reversal of E. histolytica
inhibition
0.01 0.01 0.05 0.01 0.01 0.10 1 .o 1.0 0.1 1.0
by folic acid analogues.
Compounds
Analogue
producing 100 % Inhibition, w/ml
able to reverse inhibition
Aminopterin Dichloroaminopterin
Thymidine, guanylic acid, folic acid, leucovorin, adenosine, guanosine, thymidylic acid
Oxyfolic
Folic acid, leucovorin
acid
Benzimidazole
Adenine,
9,10-dimethylpteroylglutamic acid 4-amino-lo-methylpteroylglutamic acid 9-methylpteroylglutamic acid
Folic acid, leucovorin
2,4-diamino-6(N4 sulfanilamido-S-triazine) 2-carbethoxy-4,6-diamino-S-triazine 2-carbethoxy-4-phenylamino-6-amino-S-triazine
Experimental
Cell Research 25
guanine
I None of the compounds hibition
tested reversed
in-
Antimetabolites on the growth of Entamoeba histolytica
651
aminopterin was reversed by thymidine, guanylic acid, folic acid, leucovorin, adenosine, guanosine, and thymidylic acid, but not by methionine, adenine, guanine, thymine, or vitamin B,,. This is highly suggestive that the amoebae lack the enzyme system(s) for the conversion of purine and pyrimidine bases to their corresponding nucleosides or nucleotides. It is also possible that the stage of inhibition produced by aminopterin and dichloroaminopterin is at TABLE
III.
Stimulation
of E. histolytica
Compound Cont. of compound (mg/ml) . . . Biopterin Teropterin
0 0.1 0.1
growth
by Biopterin
and Teropterin.
Amoebae per low power field 0.01 0.1 0.5 1.0
2.0
10.7 21.0
72.2 82.7
39.0 55.9
50.3 84.4
77.4 80.0
a point further along in the biosynthesis of nucleic acids than at the stage of ribosidation of the bases. Oxyfolic acid, AMPGA, DMPGA, and MPGA inhibition was reversed by folic acid and leucovorin but not by the other metabolites tested. One explanation could be that the inhibitory agents blocked more than one step thus requiring the addition of the preformed final Adenine and guanine growth factor, namely, folic acid and leucovorin. neutralized the toxic activity of benzimidazole. It was not neutralized by the other metabolites tested. Possibly the inhibitory activity of benzimidazole is due to other than blockage of folic acid synthesis or activity. None of the reversal compounds tested reduced the toxicity towards the amoebae of CDT, CPAT, and 2,4-diamino-G(N4 sulfanilamido-S-triazine). Biopterin and Teropterin were stimulatory for amoebic growth at concentrations of 0.1 to 1.0 mg/ml. The growth response of the amoebae to these two agents is recorded in Table III. Addition of adenosinetriphosphate and ribose-5-phosphate to the media containing Biopterin and Teropterin did not significantly increase amoebic numbers in the cultures. APAA was neither stimulatory nor inhibitory to E. histolytica. DISCUSSION
In view of the fact that folic acid and leucovorin are growth factors for E. histolytica, it was not surprising that aminopterin, dichloroaminopterin, oxyfolic acid, AMPGA, MPGA, and DMPGA were inhibitory to this organism. Dewey, Kidder, and Parks [2] found that Tetrahymena could be inhibited Experimental
Cell Research 25
652
M. Nakamura
by 4-amino-9-methylpteroylglutamic acid, 4-amino-lo-methylpteroylglutamic acid, and 4-amino-O,lO-dimethylpteroxylglutamic, ac.id. Folic acid released the Tetrahymena from inhibition by these compounds. Furthermore, aminopterin also reversed the activity of the antifolic which could not be demonstrated in our studies. Franklin and others [3] reported that aminopterin coli in spite of the fact that the organism does not need inhibited Escherichia folic acid for growth. Aminopterin inhibition, in this case, was reversed by thymidine, but not by folic acid, vitamin Blz, thymine, guanine, hypoxanthine, adenosine, adenylic acid, or cytidylic acid. It is possible that in microorganisms not requiring folic acid, the mechanism of action of aminopterin is at another locus since these workers found that Lactobacillus leichmanni 313 which does require folic acid can be protected from aminopterin toxicity by folic acid and thymidine. Since both aminopterin and dichloroaminopterin inhibition was overcome by thymidine, guanylic acid, thymidylic acid, adenosine, and guanosine, a possible interpretation of the data is that these agents block incorporation of both purines and pyrimidines in E. histolytica. Another strong possibility is that aminopterin and dichloroaminopterin block the activity of leucovorin, which indirectly interferes with either necessary for formate transport, utilization or interconversion of purines and pyrimidines. In some strains Nakamura [7] has found that nucleotides and nucleosides of E. histolytica, are necessary growth factors, whereas, in other strains, synthesis of these intermediates can originate de nova. The mechanism of inhibition of CDT, CPAT, and 2,4-diamino-B(N4 sulfanilamido-S-triazine) is not clear from these experiments. None of the metabolites studies reversed this inhibition. It seems likely that these chemicals are directly cytotoxic and are not involved in metabolic antagonisms. However, further experiments are required before definite conclusions can be drawn regarding these compounds. The observation that Biopterin and Teropterin were amoeba-stimulatory was not completely surprising since Broquist and Albrecht [ 1 ] showed that fasciculata, a trypanosomid Biopterin was a growth requirement for Crithidia flagellate. It is possible that Biopterin can replace folic acid as does aminopterin for Tetrahymena [2, 131. The fact that benzimidazole inhibition was not reversed by folic acid or leucovorin suggests that benzimidazole does not directly interfere with the action of folic acid but perhaps prevents purine ring closure or utilization of the purine ring. The role of adenine and guanine in releasing the amoebae from benzimidazole inhibition strengthens this concept. Experimental Cell Research 25
Antimetabolites on the growth of Entamoeba histolytica SUMMARY
Aminopterin, dichloroaminopterin, oxyfolic acid, 9,10-dimethylpteroylglutamic acid, 4-amino-lo-methylpteroylglutamic acid, and 9-methylpteroylglutamic acid inhibited Entamoeba histolytica in uitro. The inhibition was reversed by folic acid and leucovorin. 2,4-diamino-B(N4 sulfanilamido-Striazine), 2-carbethoxy-4,6-diamino-S-triazine, and 2-carbethoxy-4-phenylamino-6-amino-S-triazine were also amoebicidal but reversal of this toxicity was not possible with numerous metabolites studied. 2-amino-4-hydroxy-6(1,2,3-trihydroxypropyl-pteridine) and sodium pteroyltriglutamate stimulated growth of amoebae. Possible mechanisms of action of these chemical agents are discussed. REFERENCES 1. BROQUIST, H. P. and ALBRECHT, A. M., Proc. Sot. Exptl. Biol. Med. 89, 178 (1955). 2. DEWEY. V. C.. KIDDER, G. W. and PARKS, R. E., JR., ibid. 78. 91 (1951). 3. FRANK&N, A. i., STOK~TAD, E. L. R., HOF~MANN; C. e., BELT,‘M. aid JOKES, T. H., J. Chem. sot. 71, 3549 (1949). 4. JACOBS, L., Am. J. Hyg. 46, 172 (1947). 5. NAKAMURA, M., Proc. Sot. EzptZ. Biol. Med. 89, 680 (1955).
6. __ 7. -
ibid. 95, 524 (1957). Biol. Bull. 112, 377 (1957). 8. NAKAMURA, M. and BAKER, E. E., Am. J. Hyg. 64, 12 (1956). 9. Proc. Sot. Exptt. Biol. Med. 92, 723 (1956). 10. NAKAMURA, M. and JONSSON, S., Arch. Biochem. Biophys. 66, 183 (1957). 11. REEVES, R. E., MELENEY, H. E. and FRYE, W. W., Am. J. Hyg. 66, 56 (1957). 12. SHAFFER, J. G. and FRYE, W. W., ibid. 47, 214 (1948). 13. TITTLER, I. A. and BELSKY, M. M., Science 114, 493 (1951).
Experimental
Cell Research 25