Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Dichloro-diphenyl-trichloroethane (DDT) was extensively used in agriculture pest control and
is still used for indoor residual spraying to control malaria. The lipophylicity of DDT and its
breakdown product dichloro-diphenyl-dichloroethylene (DDE) dictates that they associate
with membranes, lipids and hydrophobic proteins in the biological environment. Their poor
degradable nature causes DDT and DDE to persist for decades in the environment and in
individuals who are or were in contact with the pesticide. In many countries the synchronised
resistance of the mosquito vector to insecticides and the malaria parasite towards antimalarial
drugs led to a drastic rise in malaria cases and to malaria epidemics. This study assesses the
influence of low level exposure of DDT and DDE on chloroquine (CQ) resistance of the dire
human malaria parasite, Plasmodium falciparum.
The in vitro activity of p,p’-DDT and p,p’-DDE towards blood stages of chloroquine sensitive
(CQS) P. falciparum D10 and chloroquine resistant (CQR) P. falciparum Dd2 was
determined using two complementary in vitro assays (Malstat and SYBR Green 1). The 50%
inhibition concentrations (IC50s) of p,p’-DDT and p,p’-DDE were found to be ±14 to 38 μM
(5-12 μg/mL) and highly similar towards CQS and CQR P. falciparum strains. This result
indicated that the proteins involved in CQ resistance have no effect on the activity of the
insecticide DDT and it breakdown product DDE.
In order to assess the influence of DDT and DDE on CQ activity, in vitro fixed ratio drug
combination assays were performed, as well as isobologram analysis. We found that CQ
works in synergy with p,p’-DDT and p,p’-DDE against CQS P. falciparum D10. However,
both p,p’-DDT and p,p’-DDE were antagonistic toward CQ activity in CQR P. falciparum
Dd2. This indicated that p,p’-DDT and p,p’-DDE do have an effect on CQ resistance or on
the action of CQ via a target other than hemozoin polymerization. The observation of
reciprocal synergism of p,p’-DDT and p,p’-DDE with CQ against CQS D10 and antagonism
against CQR Dd2 strain is highly significant and strongly indicates selection of CQ resistant
strains in the presence of p,p’-DDT and p,p’-DDE. People who have low levels of circulating
DDE and/or DDT could be at a high risk of contracting CQR malaria. However, medium term
(nine days) DDE exposure of CQS P. falciparum D10 did not induce resistance, as no
significant change in activity of CQ, p,p’-DDT and p,p’-DDE towards blood stages the CQS
strain was observed. This exposure was, however, shorter than expected for a malaria
infection and would be addressed in future studies.
From our results on the interaction of CQ with p,p’-DDT and p,p’-DDE, it was important to
assess the residual DDT and DDE variable and how much of residual p,p’-DDT and/or p,p’-
DDE would enter into or remain in the different compartments (the RPMI media, erythrocytes
and infected erythrocytes) over time. In combination with liquid-liquid extraction, we
developed a sensitive GC-MS analyses method and a novel HPLC-UV analysis method for
measuring DDT and DDE levels in malaria culturing blood and media. Whilst the HPLC-UV
method was relatively cheaper, faster, and effective in determining high DDT and DDE
concentrations, the optimised GC-MS method proved to be effective in detecting levels as
low as 78 pg/mL (ppt) DDE and 7.8 ng/mL (ppb) DDT in biological media. Using both the
HPLC and GC-MS methods we observed that malaria parasites influence distribution of the
compounds between the erythrocytic and media fractions. P. falciparum D10 infection at
±10% parasitemia lead to must faster equilibration (less than 8 hours) between compartments.
Equimolar distribution of p,p’-DDE was observed, but the parasites lead to trapping of the
largest fraction of p,p’-DDT in the erythrocyte compartment. These results indicate that a
substantial amount would reach the intra-erythrocytic parasite and could influence the
parasite directly, possibly leading to either synergistic or antagonistic drug interactions.
This study is the first to illustrate the “good and bad” of the insecticide DDT in terms of CQ
resistance and sensitivity toward the human malaria parasite P. falciparum. These results will
hopefully have an important influence on how future policies on malaria control and
treatment particularly in endemic areas will be addressed and could also have an impact on
the anti-malarial drug discovery approach. / AFRIKAANSE OPSOMMING: Dichlorodifenieltrichloroetaan (DDT) is op groot skaal in landbouplaagbeheer gebruik en
word nog steeds gebruik vir binnenshuise oppervlakbespuiting om malaria te beheer. Die
lipofilisiteit van DDT en sy afbraakproduk dichlorodifenieldichloroetileen (DDE) dikteer dat
hulle met membrane, lipiede en hidrofobiese proteïene in die biologiese omgewing
assosieer. Stadige afbraak veroorsaak dat DDT en DDE vir dekades in die omgewing
agterbly, asook in individue wat in kontak is, of was met die insekdoder. In baie lande het
gesinkroniseerde weerstand van die muskietvektor teenoor insekdoders en die malariaparasiet
teenoor antimalariamiddels gelei tot 'n drastiese styging in malariagevalle en tot malariaepidemies.
In hierdie studie word die invloed van lae vlak blootstelling van DDT en DDE op
chlorokien (CQ) weerstand van die mens malariaparasiet, Plasmodium falciparum,
geëvalueer.
Die in vitro aktiwiteit van p,p'-DDT en p,p'-DDE teenoor die bloedstadia van chlorokiensensitiewe
(CQS) P. falciparum D10 en chlorokien-weerstandbiedende (CQW) P. falciparum
Dd2 is bepaal deur gebruik te maak van twee komplementêre in vitro toetse (Malstat en
SYBR Groen toetse). Die 50% inhibisie konsentrasies (IC50s) van p,p'-DDT en p,p'-DDE is
bepaal as ±14 to 38 μM (5-12 μg/mL) en was hoogs vergelykbaar tussen CQS en CQW P.
falciparum stamme. Hierdie resultaat het aangedui dat die proteïene betrokke by CQ
weerstand geen effek op die aktiwiteit van die insekdoder DDT en die afbraakproduk DDE
het nie.
Om die invloed van DDT en DDE op CQ aktiwiteit te evalueer, is die aktiwiteit van
kombinasies van die verbindings in vaste verhoudings getoets, tesame met isobologram
ontleding. Ons het gevind dat CQ sinergisties saam met p, p'-DDT en p, p'-DDE teen CQS P.
falciparum D10 werk. Daarteenoor het beide p, p'-DDT en p, p'-DDE antagonistiese werking
getoon teenoor CQ aktiwiteit met CQW P. falciparum Dd2 as teiken. Dit het aangedui dat
p,p'-DDT en p, p'-DDE wel 'n invloed op CQ weerstand het of ‘n aktiwiteit van CQ, anders as
hemozoin polimerisasie, beïnvloed. Die waarneming van resiproke sinergisme en
antagonisme van p, p'-DDT en p, p'-DDE in kombinasie met CQ teenoor die CQS D10 en
CQW DD2 stamme respektiewelik, is hoogs betekenisvol en dui op seleksie van CQweerstandige
stamme in die teenwoordigheid van p, p'- DDT en p, p'-DDE. Mense wat lae
vlakke van sirkulerende DDE/DDT het, het dus 'n hoër risiko om CQW malaria te kry.
Verder is gevind dat medium termyn (nege dae) DDE blootstelling van CQS P. falciparum
D10 nie weerstand nie veroorsaak nie, want geen beduidende verandering in die aktiwiteit
van CQ, p,p'-DDT en p,p'-DDE teenoor die bloed stadiums van die CQS stam is waargeneem
nie. Hierdie blootstelling is egter korter as in 'n malaria-infeksie en sal verder bestudeer word
in toekomstige studies.
Vanuit die interaksie resultate van CQ met p, p'-DDT en p, p'-DDE was dit belangrik om die
residuele DDT en DDE veranderlike te evalueer, asook die distribusie van p,p'-DDT en p,p'-
DDE tussen die verskillende kompartemente (die kultuurmedium, eritrosiete en geïnfekteerde
rooibloedselle) oor verloop van tyd. In kombinasie met vloeistof-vloeistof ekstraksie, het ons
'n sensitiewe GC-MS en nuwe HPLC-UV analisemetode ontwikkel vir die meet van DDT en
DDE-vlakke in bloed (normale en geïnfekteerde eritrosiete) en die kultuurmedium. Terwyl
die HPLC-UV metode relatief goedkoper, vinniger en effektief in die bepaling van hoë DDT
en DDE-konsentrasies is, was die geoptimaliseerde GC-MS metode doeltreffend in die
opsporing van vlakke so laag as 78 pg/mL (dpt) DDE en 7.8 ng/mL (dpb) DDT in biologiese
media. Met behulp van beide die HPLC-UV en GC-MS metodes is waargeneem dat die
malariaparasiet die ekwilibrasie van die verbindings tussen die eritrosiet- en media
kompartemente beïnvloed. P. falciparum D10 infeksie met ± 10% parasitemia lei tot vinniger
ekwilibrasie (minder as 8 uur) tussen die kompartemente. Ekwimolêre verspreiding van p,p'-
DDE is waargeneem, maar die parasiete het die grooste fraksie van p,p'-DDT in die eritrosiet
kompartement vasgevang. Hierdie resultate wys dat 'n aansienlike fraksie die intraeritrositiese
parasiet kan bereik en sodoende die parasiet direk kan beïnvloed en moontlik kan
lei tot sinergistiese of antagonistiese middel interaksies.
Hierdie studie is die eerste om die "goed en sleg" van die insekdoder DDT in terme van CQ
weerstand en sensitiwiteit teenoor die menslike malariaparasiet P. falciparum te
illustreer. Hierdie resultate sal hopelik 'n belangrike invloed hê op die toekomstige beleid oor
die beheer van malaria en behandeling, veral in endemiese gebiede, en mag ook 'n impak hê
op die antimalariamiddel navorsing.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/20310 |
| Date | 03 1900 |
| Creators | Makowa, Hazel Beverly |
| Contributors | Rautenbach, Marina, Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Rights | Stellenbosh University |
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