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Title: Some aspects of resistance to arsenical drugs in Trypanosoma brucei brucei
Author: Ojeda, P. V.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1980
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1. A strain resistant to melarsen oxide was produced from a laboratory adapted line of Trypanosoma brucei using the short passage method. It was necessary to passage the original line of trypanosomes 18 times with 16 exposures to the drug to reach a resistance factor of 13. With the help of a drug sensitivity test, using the oxygen electrode and Warburg manometry, it was found that the new strain is a heterogeneous population of trypanosomes with respect to the sensitivity to melarsen oxide. The resistant character is stable after serial passage in rats. 2. Different alternatives were suggested and investigated in order to rationalise the development of resistance in biochemical terms. The alternatives were:- Modification of metabolism to bypass the action of the drug.- Modification of enzyme targets with regard to different kinetic parameters, increased specific activities and different sensitivities to the drug.- Modification of the drug transport. 3. The metabolic studies showed that although the glycolytic sequence is the basis of metabolic route in both parasites, the resistant strain shows lower pyruvate/glucose and pyruvate/oxygen ratios from glucose catabolism when compared with the parent line. Furthermore some CO is produced by the resistant strain with an RQ value of 0.1 (approx.). These characteristics are observed in the absence of the drug. The patterns of inhibition of the oxygen consumption with whole cells from the parent line and be resistant strain were different. The I5 value for the parent line was 6.25 x 10 M melarsen oxide and only 25 to 30% inhibition is observed for the same parameter at the highest concentration of drug used (250 x 10 M) in the resistant organism. The patterns of inhibition in water lysates however are apparently the same in the parent line and the resistant strain; this feature is interpreted in terms of a permeability barrier for the drug in the drug fast trypanosomes. The general carbon balance with whole cells from the parent line was severely affected under the influence of the drug as a sharp reduction was observed in the pyruvate/ glucose and pyruvate/oxygen ratios from glucose catabolism. This corroborates data already reported, suggesting that pyruvate kinase (E.C. is the focal point of action of organic arsenicals. When the same parameters were investigated in whole cells from the resistant strain a minor inhibition of the metabolism was found, with the difference that the metabolic ratios remained constant in the presence of the drug; furthermore the CO production described in the absence of the drug was abolished. Therefore it is suggested that although some common site of inhibition is still affected in the drug fast organisms, there is also the possibility of a minor pathway for the further metabolism of pyruvate when the drug is present. 4. The enzymological studies suggest that pyruvate kinase already reported as the main target of arsenicals in trypanosomes, has the same kinetic characteristics in the parent line and the resistant strain. The S50 values with respect to phosphoenolpyruvate were 1.60 - 0.30 x 1? M; Hill -coefficient = 2.16 - 0.30. The K value with respect to ADP was 3.17 - 0.22 x 10 M. The V values in terms of mg of protein were also the same in the parent and resistant line. Phosphoglycerate kinase (E.C. another target of organic arsenicals did not show any modification as a result of the development of resistance. The enzyme showed apparent hyperbolic responses with respect to ATP and phosphoglycerate; upon linear transformation of the data, ATP showed phasic linear responses with K value = 0.89 ± 0.20 x l0 M and K value = 0.30 - 01. Phosphoglycerate showed m1 a simple linear response with m2 K value = 5.80 - 0.42 10 M. The V values in terms of mg of protein were also the same. Pyruvate kinase and phosnplanxoglycerate kinase in resistant strain showed similar patterns of inhibition with respect to melarsen oxide when compared with the parent line (I. value for pyruvate kinase i = 7.60 1 (55M melarsen oxide; I50 value for phosphoglycerate kinase = 0.53 x 10 M melarsen oxide). 5. When drug transport studies were performed it was found that melarsen oxide gets into the parasite via a carrier mediated mechanism with the following kinetic parameters, calculated using a non-linear correlation: Kt value = 14.42.10 M; V = 5.19 nmoles min mg protein, Hill coefficient = 2.27. It was found that the resistant strain lost the carrier mediated transport mechanism and only a diffusion component, assumed to be present in the parent line, remains responsible for a low rate of uptake. The transport of melarsen oxide in the parent line is concentrative, inhibited by the analogue melamine (Ki 7.0.10 M), temperature dependant, inhibited by iodoacetate and possibly involves -SH groups as indicated by the lack of uptake of the pentavalent sodium melarsen.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available