Filarial glutathione S-transferase: its induction by xenobiotics and potential as drug target

Glutathione-S-transferase (GST) a Phase-II drug detoxification enzyme, was detected in Setaria cervi, a bovine filarial parasite. In vitro effect of diethylcarbamazine, butylated hydroxyanisole and phenobarbitone on the GST of adult female S. cervi was assayed by the addition of these compounds in the maintenance medium. The specific activity of GST towards 1-chloro-2,4-dinitrobenzene was increased progressively 1.2-1.97, 1.3-2.4 and 1.2-2.7 times at 10-100 µM of diethylcarbamazine, butylated hydroxyanisole and phenobarbitone, respectively, after 5 h at 37°C. Substrate specificity studies showed a higher increase in specific activity with ethacrynic acid and no change with cumene hydroperoxide. Although the intensity of GST activity band was more in extract from diethylcarbamazine or butylated hydroxyanisole treated worms extract, an extra band of activity appeared in those worm extracts compared to control worm extract. SDS/PAGE showed increased thickness of the band corresponding to purified GST in extracts from diethylcarbamazine/butylated hydroxyanisole/phenobarbitone treated worms. Purification and quantification of GST from diethylcarbamazine and butylated hydroxyanisole treated worms indicated an increase in enzyme specific activity. The increase in GST protein by these agents was blocked by prior treatment with actinomycin D, indicative of a transcription dependent response. The role of this enzyme in motility and viability of microfilariae and adult female was tested in vitro using a range of known GST inhibitors. Of those tested, ethacrynic acid, ellagic acid, 1-chloro-2,4-dinitrobenzene, cibacron blue and butylated hydroxyanisole reduced the viability and motility of microfilariae and adult female worms at micromolar concentrations. These results suggest that S. cervi GST is inducible in response to the antifilarial drug diethylcarbamazine and may play an important role in parasite's survival, thus could be a potential drug target

The effect of E-64 on total reactive oxygen species production in parasites.

<p>Adult female parasites were exposed to 5, 10, 20 and 40 μM E-64 for 8 h in the KRB maintenance medium. Total Reactive oxygen species generated was measured using NBT as substrate. The data expressed is Mean ± SEM of at least three values (n = 3). ^***P<0.0001, ^**P<0.001, ^*P<0.05. Values with P<0.05 were considered significant.</p

Interaction of E-64 with GSH was drawn using ChemDraw Ultra 7.0.

<p>(A) <i>In vitro</i> effect of E-64 on GSH using DTNB as substrate (B) Possible interaction of E-64 with GSH molecule.</p

The effect of E-64 on viability of adult parasites and microfilariae.

<p>(A) Adult female worms (n = 10) of equal size were incubated in 20 ml maintenance at specified experimental conditions and viability was assessed by MTT assay after 8 h of exposure. Worms incubated in maintenance medium only served as control. (B) Microfilariae (mf) recovered from the uterus of gravid female of control and treated set. Adult female parasites were dissected longitudinally and the released mf was visualized under microscope at 40× (Motic B1 series). Data expressed is Mean ± SEM of at least three values (n = 3). ^***P<0.0001, ^**P<0.001, ^*P<0.05. Values with P<0.05 were considered significant.</p

IC₅₀ of E-64 for cathepsin B activity.

<p>The half maximal inhibitory concentration (IC₅₀) was calculated by plotting the graph between the different concentration of E-64 and the % inhibition in cathepsin B activity.</p

Effect of E-64 on the motility of adult female <i>S. cervi</i> parasite.

<p>The motility of the parasites was visually checked at different time intervals. Adult female worms (n = 10) of equal size were incubated with 5, 10, 20 and 40 μM concentration of E-64 in 20 ml maintenance medium at 37°C and 5% CO₂ for 8 h. Worms incubated in only maintenance medium served as Control. Worm motility was scored as -, no movement; +, least active; ++, less active; +++, moderately active; and ++++, highly active. ©Worms were transferred into fresh medium (devoid of E-64) after 8 h and motility recovery in treated group was compared to control group. Results are from three independent experiments performed in duplicates.</p

The protein content of the control and E-64 treated parasites.

<p>The protein content was determined using Bradford assay using BSA as standard. Data expressed is Mean ± SEM of at least three values (n = 3). ^***P<0.0001, ^**P<0.001, ^*P<0.05. Values with P<0.05 were considered significant.</p