185 tase.
This conclusion accords with results in both
nor-
mal rodent brain tissue and brain tumours. 14,15 Brain
tumours
generally
contained less
dihydrofolate
reductase than other human tissues assayed by us. This could be the result of prolonged storage, the fact that brain dihydrofolate reductase is more labile than that from other tissues,2 or the fact that these tumours only contain small amounts of enzyme. The level of human enzyme activity is about 10% that found in normal rabbit brain.2 The presence of dihydrofolate-reductase activity in human brain tumours provides a biochemical rationale for therapy with antifolates. Lipid-soluble antifolates would seem to offer a particular advantage because of their increased penetration into the cerebospinal fluid
(c.s.F.)’6 treatment of c.N.s. tumours has had of varying degrees success .4,1,11-20 Dihydrofolate reductase is the putative cytotoxic target of methotrexate and other antifolates. Methotrexate binds with high affinity to the enzyme and thus inhibits the formation of tetrahydrofolate and consequently the de novo biosynthesis of purine and thymidine.21,22 In addition, methotrexate may limit catecholamine and serotonin biosynthesis in the c.N.S. The mechanism for this inhibition involves the dihydrofolate-reductase-catalysed reduction of 7, 8-
Methotrexate
dihydrobiopterin to tetrahydrobiopterin.23 Tetrahydrobiopterin is a cofactor in the hydroxylation of phenylalanine, tyrosine, and tryptophan, which leads to the formation of biogenic amines and serotonin.24,25 A biochemical basis for the C.N.S. toxicity of methotrexate therapy may, therefore, be related to the inhibition of synthesis of tetrahydrofolate or tetrahydrobiopterin or both. The
of dihydrofolate reductase in brain tumours may accelerate progress towards a selective approach to their therapy. Conventional and high-dose methotrexate treatment have already been tried in advanced cases after other therapy has been unsuccessful. With a clear rationale for its use, however, methotrexate and other antifolates might be considered as a first-line treatment. The selectivity of antifolates might be accentuated by other biochemical manipulations. Probenecid reduces the efflux of methotrexate from the c.s.F. in rabbits26 and carboxypeptidase G1 will cleave the glutamate moiety from methotrexate rendering it inactive and thereby decreasing the effective plasma concentration.2’ These points might be exploited by use of either high-dose methotrexate or intrathecal methotrexate with probenecid to maintain c.s.F. levels and carboxypeptidase G to prevent peripheral toxicity.
finding
We thank Dr G. P. Beardsley for helpful discussions. This study was m part by Public Health Service grant CA 18662. Herbert T. Abelson is the recipient of Research Career Development Award
supported
CA00075.
Requests for reprints should be addressed to: H.T.A., Sidney Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, U.S.A.
REFERENCES 1
Makulu,
D.
R., Smith, E. F., Bertino, J. R. J. Neurochem. 1973, 21, 241. 2. Spector, R., Levy, P., Abelson, H. T. Biochem. Pharmac. 1977, 26, 1507. 3. Spector, R., Levy, P., Abelson, H. T. J. Neurochem. 1977, 29, 919. 4. Bleyer, W. A., Drake, J. C., Chabner, B. A. New Engl. J. Med. 1973, 189, 773.
PERSISTENCE OF DRUG-RESISTANT MALARIA PARASITES R. HALL G. H. BEALE
V. E. ROSARIO D. WALLIKER
Protozoan Genetics Unit, Institute of Animal Genetics, West Mains Road, Edinburgh EH9 3JN
Mixtures of drug-resistant and sensitive forms of Plasmodium chabaudi were used to infect mice, and the resulting infections were maintained in the absence of drug. Both chloroquineresistant and pyrimethamine-resistant mutants were able to survive in such mixed infections. The results of experiments on chloroquine resistance indicated an apparent selective advantage of the resistant over the sensitive form.
Summary
INTRODUCTION
IN the absence of any satisfactory vaccine, chemotherapy is the only effective treatment of malaria. Hence, the development of drug-resistant forms of malaria parasites, which is impeding the control of the disease in many parts of the world,I,2 is viewed with concern. Resistance of Plasmodium falciparum to chloroquine has spread rapidly in recent years, especially in South-East Asia, and has severely limited the effectiveness of this drug over wide areas. Resistance to other widely used antimalarial drugs, such as pyrimethamine, has also appeared, and there is concern that resistance to newly developed drugs such as mefloquine3 might develop and spread in the same way. A little-studied aspect of drug resistance in malaria parasites is the capacity of the resistant forms to persist in the absence of the drug. This would become particularly important if use of a given drug were to be discontinued for a period in the hope that the resistant forms
Bishop, Y., Jaffe, N., Furman, L., Traggis, D., Frei, E. Blood, 1975, 45, 189. 6. Shapiro, W. R., Chernik, N. L., Posner, J. B. Archs Neurol. 1973, 28, 96. 7. Bresnan, M. J., Gilles, F. H., Lorenzo, A. V., Walters, G. V., Barlow, C. F. Trans. Am. Neurol. Ass. 1972, 97, 204. 8. Meadows, A. T., Evans, A. E. Cancer, 1976, 37, 1079. 9. Rubinstein, L. J., Herman, M. M., Long, T. F. et al, Cancer, 1975, 35, 291. 10. Perkins, J. P., Hillcoat, B. L., Bertino, J. R. J. biol. Chem. 1967, 242, 4771. 11. Rothenberg, S. P. Analyt. Biochem. 1965, 13, 530. 12. Kamen, B. A., Takach, P. L., Vatev, R., Caston, J. D. ibid. 1976, 70, 54. 13. Lowrey, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. J. biol. Chem. 1951, 193, 265. 14. Levin, V. A., Clancy, T. P., Ausman, J. I., Rall, D. P. J. natn. Cancer Inst. 1972, 48, 875. 15. Duch, D. S., Bowers, S. W., Bigner, D. D., and Nichol, C. A. Proc. Am. Ass. Cancer Res. 1977, 18, 177 (abstr.). 16. Nichol, C. A., Cavallito, J. C., Woolley, J. L., Sigel, C. W. Cancer Treat. Rep. 1977, 61, 599. 17. Djerassi, I., Kim, J. S., Shulman, K. ibid. p. 691. 18 Rosen, G., Ghavimi, F., Nirenberg, A., Mosende, C., Mehta, B. M. ibid. p. 681. 19. Shapiro, W., R. ibid. p. 753. 20. Tator, C. H. Cancer Res. 1976, 36, 3058. 21. Hryniuk, W. M. ibid. 1975, 35, 1085. 22. Goldman, I. D. Mol. Pharmac 1974, 10, 257. 23. Spector, R., Fosburg, M., Levy, P., Abelson, H. T. J. Neurochem. (in the press). 24. Friedman, P. A., Kappelman, A. H., Kaufman, S. J. biol. Chem. 1972, 247, 5. Geiser, C. F.,
4165. 25. Kaufman, S. in Frontiers in Catecholamine Research (edited by E. Usdin and S. H. Snyder); p. 53. Oxford, 1973. 26. Spector, R. Cancer Treat. Rep. 1976, 60, 913. 27. Chabner, B. A., Johns, D. G., Bertino, J. R. Nature, 1972, 239, 395.
186 revert to, or be displaced by, sensitive ones. This be might expected to happen, in view of the finding in research that mutants are on the whole less fit genetic than the wild-type strains. We have investigated the persistence of drug-resistant forms of the rodent malaria parasite P. chabaudi. We have shown that chloroquine-resistant and pyrimethamine-resistant forms can be obtained by selection in the presence of these drugs.4,s Resistance is inherited in a stable manner through blood and mosquito transmission, and it shows mendelian recombination and segregation with other genetic markers when resistant forms are crossed with sensitive forms. It can thus be considered to have arisen by spontaneous gene mutation and subsequent selection under drug pressure. In the experiments described here, we constructed mixed populations of resistant and sensitive parasites, maintained the populations for various periods in the blood of mice without drug administration, and later took samples to determine the proportion of resistant and sensitive forms remaining. In each experiment, resistant forms were mixed with representatives of the parent sensitive line from which they had derived, thus ensuring that the lines compared were genetically alike except for the resistance character under study.
TABLE I--CHARACTERISTICS OF PARENT LINES
would
METHODS
Three P. chabaudi lines were used (table I). Line 1 (drugsensitive) was a clone derived from parasites of a thicket-rat (Thamnomys rutilans) from Central Africa. Line 2 (pyrimethamine-resistant) was derived from line 1 by single-step treatment of blood forms with a high dose of pyrimethamine (50 mg/kg mouse body-weight for 4 days),4 and line 3 (resistant to pyrimethamine and chloroquine) was derived from line 2 by continuous treatment of blood forms with doses of chloroquine (2 mg/kg and 3 mg/kg) during five mouse passages.s TABLE
II——CHLOROQUINE-RESISTANT/SENSITIVE MIXTURE:
III-PYRIMETHAMINE-RESISTANT/SENSITIVE
than 3 days. Clones were tested for pyrimethamine resistance by injection of 106 blood forms into three mice which were then treated with pyrimethamine at 15 mg/kg for 4 days. This treatment eradicated line 1 but not lines 2 and 3. Chloroquine resistance was tested similarly, with doses of 3 mg/kg daily for 6 days, which eradicated lines 1 and 2 but not 3. Control infections of each line alone were cloned and examined for drug response at similar times to the mixtures. RESULTS
Mixtures of Chloroquine Resistant and Sensitive Parasites Four mixtures were made of lines 2 and 3, in proportions of 50:50 (experiments 1 and 2), 10:90 (experiment 3), and 90:10 (experiment 4). The results are shown in table n. In experiment 1, where cloning was carried out at three stages, both sensitive and resistant parasites
NUMBER OF CLONES ISOLATED AND RESPONSE TO
CLONES
TABLE
In each experiment mixtures of 1 x 106 blood forms containing known proportions of resistant and sensitive parasites were injected into young female C57 black mice. At various times samples of blood were taken and clones of blood forms were established with a dilution method.4 The actual days of cloning differed in the different experiments, but clones were usually prepared shortly after inoculation of the mixture (day 3), soon after the first peak ofparasitaemia (day 10 or 11), and again on a day when a recrudescence of parasitaemia occurred. Such recrudescences usually took place between days 26 and 36, when the parasitaemia rarely rose above 5% and lasted no more
CHLOROQUINE
(-=NO
ISOLATED)
MIXTURES: NUMBERS OF CLONES ISOLATED AND RESPONSE TO PYRIMETHAMINE
187 on day 3, but only resistant forms were recrudescent parasitoemia. In experithe found during and 4 ments 2, 3, cloning was carried out only at recruinstance all clones isolated were and in each descence, resistant.
were
detected
Mixtures of
Pyrtmethamine Resistant and Sensitive
Parasites Three experiments were carried out with mixtures of equal numbers of lines 1 and 2 (table III). In experiments 1 and 3 both resistant and sensitive forms were detected at early stages of the infections, but all clones (total 48) derived from the recrudescent parasitxmias were sensitive. In experiment 2, on the other hand, resistant as well as sensitive clones were detected at all stages. Control Experiments with Unmixed Lines Tables II and III also show the numbers of sensitive and drug-resistant clones isolated from infections produced by lines 1, 2, and 3 alone. Each clone shows the drug response of its uncloned parent line at all stages. Hence, under these conditions the lines appeared to be quite stable in their resistance characteristics.
This work was
was
supported by
tant.
The results for pyrimethamine resistance are less in two of the three mixed infections the sensitive form appeared to outgrow the resistant, while in the third both forms grew equally well. Larger numbers of clones would have to be isolated to give significant
striking;
a
Requests for repi:ints should be addressed to D. W. REFERENCES 1. Tech. Rep. Ser. Wld Hlth Org. 1973, 529, 1. 2. Peters, W. Adv. Parasit. 1974, 12, 69. 3. Trenholm, G. M., Williams, R. L., Desjardins, R. E., Frischer, H., P. E., Rieckmann, K. H., Canfield, C. J. Science, 1975, 190, 792. 4. Walliker, D., Carter, R., Sanderson, A. Parasitology, 1975, 70, 19. 5. Rosario, V. E. Nature, 1976, 261, 585. 6. Clyde, D. F. Malaria in Tanzania. London, 1967.
Carson,
Hypothesis SMOKING AND INDUSTRIAL POLLUTION, AND THEIR EFFECTS ON MENOPAUSE AND OVARIAN CANCER DONALD R. MATTISON
Department of Obstetrics and Gynecology, Columbia Presbyterian Medical Center, New York, New York 10032, U.S.A. SNORRI S. THORGEIRSSON
DISCUSSION
The results show an unexpected preferential (or exclusive) survival of chloroquine-resistant over sensitive forms-the opposite of what had been expected on a basis of selective disadvantage of mutants. Even where the initial proportion of resistant/sensitive parasites was 10:90 (experiment 4, table 11), all the clones derived from the recrudescent parasites were chloroquine-resis-
the Medical Research Council. V. E. R. grant from the Calouste Gulbenkian Foundation.
supported by
Laboratory of Chemical Pharmacology, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. The rodent ovary contains an enzyme system(s) capable of metabolising polyaromatic hydrocarbons to reactive electrophilic incyclic termediates known to cause cytotoxicity, mutation, and cancer. If the human ovary contains similar enzyme systems, metabolic activation of environmental chemicals could explain the earlier menopause in cigarette smokers and the higher incidence of ovarian cancer in industrialised areas.
Summary
results.
Although no experimental work of the type described here has been carried out with malaria parasites infecting man, there is some epidemiological evidence that drug-resistant forms, once they have arisen, can survive and spread in the absence of further drug treatment. Clyde6 found pyrimethamine-resistant P. falciparum in regions of Tanzania several years after large-scale use of the drug had ended. Further, there was evidence that the resistant form had spread from its presumed place of origin into surrounding areas in which no pyrimethamine was thought to have been used. Similar studies have not been made for chloroquine resistance. As already mentioned, chloroquine-resistant forms of P. falparum have developed and spread rapidly throughout parasite populations in South-East Asia in recent years, although this has probably been assisted by continued widespread use of chloroquine in these regions. A more detailed account of these experiments, including some studies of transmission of resistant forms through mosquitoes, will be published elsewhere. The results so far obtained, though based on small numbers, show that the introduction of drug treatment against malaria parasites in a given area may result in the establishment of drug-resistant parasites in that area which persist after the drug is suspended. In such cases, drug withdrawal might not result in a reversion to sensitivity.
adversely affected by various environmental, occupational, or iatrogenic factors. The incidence of ovarian cancer is highest in industrialised urban regions, and women working in the rubber, elec-
THE ovary may be
Fig. 1—Oocyte destruction after benzo(a)pyrene treatment. Weanling C57BL/6N mice were given a single intraperitoneal injection of benzo(a)pyrene dissolved in corn oil five days before being killed. Ovaries
where.9
were
prepared and oocytes counted
as
described else-
This conclusion accords with results in both
nor-
mal rodent brain tissue and brain tumours. 14,15 Brain
tumours
generally
contained less
dihydrofolate
reductase than other human tissues assayed by us. This could be the result of prolonged storage, the fact that brain dihydrofolate reductase is more labile than that from other tissues,2 or the fact that these tumours only contain small amounts of enzyme. The level of human enzyme activity is about 10% that found in normal rabbit brain.2 The presence of dihydrofolate-reductase activity in human brain tumours provides a biochemical rationale for therapy with antifolates. Lipid-soluble antifolates would seem to offer a particular advantage because of their increased penetration into the cerebospinal fluid
(c.s.F.)’6 treatment of c.N.s. tumours has had of varying degrees success .4,1,11-20 Dihydrofolate reductase is the putative cytotoxic target of methotrexate and other antifolates. Methotrexate binds with high affinity to the enzyme and thus inhibits the formation of tetrahydrofolate and consequently the de novo biosynthesis of purine and thymidine.21,22 In addition, methotrexate may limit catecholamine and serotonin biosynthesis in the c.N.S. The mechanism for this inhibition involves the dihydrofolate-reductase-catalysed reduction of 7, 8-
Methotrexate
dihydrobiopterin to tetrahydrobiopterin.23 Tetrahydrobiopterin is a cofactor in the hydroxylation of phenylalanine, tyrosine, and tryptophan, which leads to the formation of biogenic amines and serotonin.24,25 A biochemical basis for the C.N.S. toxicity of methotrexate therapy may, therefore, be related to the inhibition of synthesis of tetrahydrofolate or tetrahydrobiopterin or both. The
of dihydrofolate reductase in brain tumours may accelerate progress towards a selective approach to their therapy. Conventional and high-dose methotrexate treatment have already been tried in advanced cases after other therapy has been unsuccessful. With a clear rationale for its use, however, methotrexate and other antifolates might be considered as a first-line treatment. The selectivity of antifolates might be accentuated by other biochemical manipulations. Probenecid reduces the efflux of methotrexate from the c.s.F. in rabbits26 and carboxypeptidase G1 will cleave the glutamate moiety from methotrexate rendering it inactive and thereby decreasing the effective plasma concentration.2’ These points might be exploited by use of either high-dose methotrexate or intrathecal methotrexate with probenecid to maintain c.s.F. levels and carboxypeptidase G to prevent peripheral toxicity.
finding
We thank Dr G. P. Beardsley for helpful discussions. This study was m part by Public Health Service grant CA 18662. Herbert T. Abelson is the recipient of Research Career Development Award
supported
CA00075.
Requests for reprints should be addressed to: H.T.A., Sidney Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, U.S.A.
REFERENCES 1
Makulu,
D.
R., Smith, E. F., Bertino, J. R. J. Neurochem. 1973, 21, 241. 2. Spector, R., Levy, P., Abelson, H. T. Biochem. Pharmac. 1977, 26, 1507. 3. Spector, R., Levy, P., Abelson, H. T. J. Neurochem. 1977, 29, 919. 4. Bleyer, W. A., Drake, J. C., Chabner, B. A. New Engl. J. Med. 1973, 189, 773.
PERSISTENCE OF DRUG-RESISTANT MALARIA PARASITES R. HALL G. H. BEALE
V. E. ROSARIO D. WALLIKER
Protozoan Genetics Unit, Institute of Animal Genetics, West Mains Road, Edinburgh EH9 3JN
Mixtures of drug-resistant and sensitive forms of Plasmodium chabaudi were used to infect mice, and the resulting infections were maintained in the absence of drug. Both chloroquineresistant and pyrimethamine-resistant mutants were able to survive in such mixed infections. The results of experiments on chloroquine resistance indicated an apparent selective advantage of the resistant over the sensitive form.
Summary
INTRODUCTION
IN the absence of any satisfactory vaccine, chemotherapy is the only effective treatment of malaria. Hence, the development of drug-resistant forms of malaria parasites, which is impeding the control of the disease in many parts of the world,I,2 is viewed with concern. Resistance of Plasmodium falciparum to chloroquine has spread rapidly in recent years, especially in South-East Asia, and has severely limited the effectiveness of this drug over wide areas. Resistance to other widely used antimalarial drugs, such as pyrimethamine, has also appeared, and there is concern that resistance to newly developed drugs such as mefloquine3 might develop and spread in the same way. A little-studied aspect of drug resistance in malaria parasites is the capacity of the resistant forms to persist in the absence of the drug. This would become particularly important if use of a given drug were to be discontinued for a period in the hope that the resistant forms
Bishop, Y., Jaffe, N., Furman, L., Traggis, D., Frei, E. Blood, 1975, 45, 189. 6. Shapiro, W. R., Chernik, N. L., Posner, J. B. Archs Neurol. 1973, 28, 96. 7. Bresnan, M. J., Gilles, F. H., Lorenzo, A. V., Walters, G. V., Barlow, C. F. Trans. Am. Neurol. Ass. 1972, 97, 204. 8. Meadows, A. T., Evans, A. E. Cancer, 1976, 37, 1079. 9. Rubinstein, L. J., Herman, M. M., Long, T. F. et al, Cancer, 1975, 35, 291. 10. Perkins, J. P., Hillcoat, B. L., Bertino, J. R. J. biol. Chem. 1967, 242, 4771. 11. Rothenberg, S. P. Analyt. Biochem. 1965, 13, 530. 12. Kamen, B. A., Takach, P. L., Vatev, R., Caston, J. D. ibid. 1976, 70, 54. 13. Lowrey, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. J. biol. Chem. 1951, 193, 265. 14. Levin, V. A., Clancy, T. P., Ausman, J. I., Rall, D. P. J. natn. Cancer Inst. 1972, 48, 875. 15. Duch, D. S., Bowers, S. W., Bigner, D. D., and Nichol, C. A. Proc. Am. Ass. Cancer Res. 1977, 18, 177 (abstr.). 16. Nichol, C. A., Cavallito, J. C., Woolley, J. L., Sigel, C. W. Cancer Treat. Rep. 1977, 61, 599. 17. Djerassi, I., Kim, J. S., Shulman, K. ibid. p. 691. 18 Rosen, G., Ghavimi, F., Nirenberg, A., Mosende, C., Mehta, B. M. ibid. p. 681. 19. Shapiro, W., R. ibid. p. 753. 20. Tator, C. H. Cancer Res. 1976, 36, 3058. 21. Hryniuk, W. M. ibid. 1975, 35, 1085. 22. Goldman, I. D. Mol. Pharmac 1974, 10, 257. 23. Spector, R., Fosburg, M., Levy, P., Abelson, H. T. J. Neurochem. (in the press). 24. Friedman, P. A., Kappelman, A. H., Kaufman, S. J. biol. Chem. 1972, 247, 5. Geiser, C. F.,
4165. 25. Kaufman, S. in Frontiers in Catecholamine Research (edited by E. Usdin and S. H. Snyder); p. 53. Oxford, 1973. 26. Spector, R. Cancer Treat. Rep. 1976, 60, 913. 27. Chabner, B. A., Johns, D. G., Bertino, J. R. Nature, 1972, 239, 395.
186 revert to, or be displaced by, sensitive ones. This be might expected to happen, in view of the finding in research that mutants are on the whole less fit genetic than the wild-type strains. We have investigated the persistence of drug-resistant forms of the rodent malaria parasite P. chabaudi. We have shown that chloroquine-resistant and pyrimethamine-resistant forms can be obtained by selection in the presence of these drugs.4,s Resistance is inherited in a stable manner through blood and mosquito transmission, and it shows mendelian recombination and segregation with other genetic markers when resistant forms are crossed with sensitive forms. It can thus be considered to have arisen by spontaneous gene mutation and subsequent selection under drug pressure. In the experiments described here, we constructed mixed populations of resistant and sensitive parasites, maintained the populations for various periods in the blood of mice without drug administration, and later took samples to determine the proportion of resistant and sensitive forms remaining. In each experiment, resistant forms were mixed with representatives of the parent sensitive line from which they had derived, thus ensuring that the lines compared were genetically alike except for the resistance character under study.
TABLE I--CHARACTERISTICS OF PARENT LINES
would
METHODS
Three P. chabaudi lines were used (table I). Line 1 (drugsensitive) was a clone derived from parasites of a thicket-rat (Thamnomys rutilans) from Central Africa. Line 2 (pyrimethamine-resistant) was derived from line 1 by single-step treatment of blood forms with a high dose of pyrimethamine (50 mg/kg mouse body-weight for 4 days),4 and line 3 (resistant to pyrimethamine and chloroquine) was derived from line 2 by continuous treatment of blood forms with doses of chloroquine (2 mg/kg and 3 mg/kg) during five mouse passages.s TABLE
II——CHLOROQUINE-RESISTANT/SENSITIVE MIXTURE:
III-PYRIMETHAMINE-RESISTANT/SENSITIVE
than 3 days. Clones were tested for pyrimethamine resistance by injection of 106 blood forms into three mice which were then treated with pyrimethamine at 15 mg/kg for 4 days. This treatment eradicated line 1 but not lines 2 and 3. Chloroquine resistance was tested similarly, with doses of 3 mg/kg daily for 6 days, which eradicated lines 1 and 2 but not 3. Control infections of each line alone were cloned and examined for drug response at similar times to the mixtures. RESULTS
Mixtures of Chloroquine Resistant and Sensitive Parasites Four mixtures were made of lines 2 and 3, in proportions of 50:50 (experiments 1 and 2), 10:90 (experiment 3), and 90:10 (experiment 4). The results are shown in table n. In experiment 1, where cloning was carried out at three stages, both sensitive and resistant parasites
NUMBER OF CLONES ISOLATED AND RESPONSE TO
CLONES
TABLE
In each experiment mixtures of 1 x 106 blood forms containing known proportions of resistant and sensitive parasites were injected into young female C57 black mice. At various times samples of blood were taken and clones of blood forms were established with a dilution method.4 The actual days of cloning differed in the different experiments, but clones were usually prepared shortly after inoculation of the mixture (day 3), soon after the first peak ofparasitaemia (day 10 or 11), and again on a day when a recrudescence of parasitaemia occurred. Such recrudescences usually took place between days 26 and 36, when the parasitaemia rarely rose above 5% and lasted no more
CHLOROQUINE
(-=NO
ISOLATED)
MIXTURES: NUMBERS OF CLONES ISOLATED AND RESPONSE TO PYRIMETHAMINE
187 on day 3, but only resistant forms were recrudescent parasitoemia. In experithe found during and 4 ments 2, 3, cloning was carried out only at recruinstance all clones isolated were and in each descence, resistant.
were
detected
Mixtures of
Pyrtmethamine Resistant and Sensitive
Parasites Three experiments were carried out with mixtures of equal numbers of lines 1 and 2 (table III). In experiments 1 and 3 both resistant and sensitive forms were detected at early stages of the infections, but all clones (total 48) derived from the recrudescent parasitxmias were sensitive. In experiment 2, on the other hand, resistant as well as sensitive clones were detected at all stages. Control Experiments with Unmixed Lines Tables II and III also show the numbers of sensitive and drug-resistant clones isolated from infections produced by lines 1, 2, and 3 alone. Each clone shows the drug response of its uncloned parent line at all stages. Hence, under these conditions the lines appeared to be quite stable in their resistance characteristics.
This work was
was
supported by
tant.
The results for pyrimethamine resistance are less in two of the three mixed infections the sensitive form appeared to outgrow the resistant, while in the third both forms grew equally well. Larger numbers of clones would have to be isolated to give significant
striking;
a
Requests for repi:ints should be addressed to D. W. REFERENCES 1. Tech. Rep. Ser. Wld Hlth Org. 1973, 529, 1. 2. Peters, W. Adv. Parasit. 1974, 12, 69. 3. Trenholm, G. M., Williams, R. L., Desjardins, R. E., Frischer, H., P. E., Rieckmann, K. H., Canfield, C. J. Science, 1975, 190, 792. 4. Walliker, D., Carter, R., Sanderson, A. Parasitology, 1975, 70, 19. 5. Rosario, V. E. Nature, 1976, 261, 585. 6. Clyde, D. F. Malaria in Tanzania. London, 1967.
Carson,
Hypothesis SMOKING AND INDUSTRIAL POLLUTION, AND THEIR EFFECTS ON MENOPAUSE AND OVARIAN CANCER DONALD R. MATTISON
Department of Obstetrics and Gynecology, Columbia Presbyterian Medical Center, New York, New York 10032, U.S.A. SNORRI S. THORGEIRSSON
DISCUSSION
The results show an unexpected preferential (or exclusive) survival of chloroquine-resistant over sensitive forms-the opposite of what had been expected on a basis of selective disadvantage of mutants. Even where the initial proportion of resistant/sensitive parasites was 10:90 (experiment 4, table 11), all the clones derived from the recrudescent parasites were chloroquine-resis-
the Medical Research Council. V. E. R. grant from the Calouste Gulbenkian Foundation.
supported by
Laboratory of Chemical Pharmacology, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. The rodent ovary contains an enzyme system(s) capable of metabolising polyaromatic hydrocarbons to reactive electrophilic incyclic termediates known to cause cytotoxicity, mutation, and cancer. If the human ovary contains similar enzyme systems, metabolic activation of environmental chemicals could explain the earlier menopause in cigarette smokers and the higher incidence of ovarian cancer in industrialised areas.
Summary
results.
Although no experimental work of the type described here has been carried out with malaria parasites infecting man, there is some epidemiological evidence that drug-resistant forms, once they have arisen, can survive and spread in the absence of further drug treatment. Clyde6 found pyrimethamine-resistant P. falciparum in regions of Tanzania several years after large-scale use of the drug had ended. Further, there was evidence that the resistant form had spread from its presumed place of origin into surrounding areas in which no pyrimethamine was thought to have been used. Similar studies have not been made for chloroquine resistance. As already mentioned, chloroquine-resistant forms of P. falparum have developed and spread rapidly throughout parasite populations in South-East Asia in recent years, although this has probably been assisted by continued widespread use of chloroquine in these regions. A more detailed account of these experiments, including some studies of transmission of resistant forms through mosquitoes, will be published elsewhere. The results so far obtained, though based on small numbers, show that the introduction of drug treatment against malaria parasites in a given area may result in the establishment of drug-resistant parasites in that area which persist after the drug is suspended. In such cases, drug withdrawal might not result in a reversion to sensitivity.
adversely affected by various environmental, occupational, or iatrogenic factors. The incidence of ovarian cancer is highest in industrialised urban regions, and women working in the rubber, elec-
THE ovary may be
Fig. 1—Oocyte destruction after benzo(a)pyrene treatment. Weanling C57BL/6N mice were given a single intraperitoneal injection of benzo(a)pyrene dissolved in corn oil five days before being killed. Ovaries
where.9
were
prepared and oocytes counted
as
described else-
