M. Meaney

A scanning electron microscope study on the route of entry of triclabendazole into the liver fluke, Fasciola hepatica.

SUMMARY Studies have been carried out to establish the relative importance of oral and trans-tegumental uptake of triclabendazole by the liver fluke, Fasciola hepatica. Experiments were designed to block either oral uptake of drug, by use of ligatures, or trans-tegumental diffusion, by allowing the drug to bind to bovine serum albumin (BSA) in the medium. Changes to the surface morphology of the tegument and gut were assessed by scanning electron microscopy. Flukes were incubated in vitro for 24 h in TCBZ.SO at a concentration of 15 microg/ml. Tegumental disruption in ligatured and non-ligatured flukes was similar, suggesting that closing the oral route did not affect drug uptake. The gut remained unaffected by drug treatment. When BSA (30 mg/ml) was present in the medium, there was a marked decline in the level of tegumental disruption. Again, the gut retained a normal morphology. Non-ligatured flukes were also incubated for 24 h in vitro in TCBZ.SO (15 microg/ml) in the presence of red blood cells. Oral ingestion of blood was demonstrated, although the gut surface retained a normal morphology. In contrast, the tegumental surface was severely affected by the drug. The findings support previous pharmacological studies which suggest that trans-tegumental uptake of triclabendazole predominates in the liver fluke.

A transmission electron microscope study on the route of entry of triclabendazole into the liver fluke, Fasciola hepatica

Uptake of triclabendazole by the liver fluke, Fasciola hepatica has been studied by experiments designed to block either oral uptake of drug, by use of ligatures, or trans-tegumental diffusion, by allowing the drug to bind to an excess of bovine serum albumin (BSA) in the medium. Changes to the tegumental system, musculature and gut were assessed using transmission electron microscopy. Flukes were incubated in vitro for 24 h in TCBZ.SO (15 microg/ml). Disruption to the tegument and muscle was similar in ligatured and non-ligatured flukes, suggesting that closing the oral route did not affect drug uptake. The ultrastructure of the gastrodermal cells remained unchanged. Non-ligatured flukes were also incubated for 24 h in vitro in TCBZ.SO (15 microg/ml) in the presence of red blood cells (RBCs). Oral uptake of blood was demonstrated, but gut ultrastructure remained normal, whereas the tegument was severely disrupted. In separate experiments, ligatured and non-ligatured flukes were incubated in TCBZ.SO (15 microg/ml) in the presence of BSA (30 mg/ml) for 24 h in vitro. There was a marked decrease in the degree of tegumental disruption observed compared with TCBZ.SO action alone; again, the gut remained normal. The findings support previous morphological and pharmacological studies indicating that trans-tegumental uptake of triclabendazole predominates in the liver fluke.

Increased action of triclabendazole (TCBZ) in vitro against a TCBZ-resistant isolate of Fasciola hepatica following its co-incubation with the P-glycoprotein inhibitor, R(+)-verapamil

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. In the first experiment, flukes were initially incubated for 2h in R(+)-VPL (1×10(-4) M), then incubated in R(+)-VPL + triclabendazole sulphoxide (TCBZ.SO) (50μg/ml) until flukes ceased movement (at 9h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15h). In the third experiment, flukes were incubated for 24h in R(+)-VPL on its own. Changes to the tegumental system and gut following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the tegumental syncytium and tegumental cells; the changes were consistent with a stress response by the fluke to drug action. Greater disruption was observed when the drugs were combined, in terms of the vacuolation and sloughing of the syncytium, spine disruption and the cessation of secretory activity in, and degradation of, the tegumental cells. In the gut, treatment with R(+)-VPL on its own did not lead to any cellular changes. Some limited changes to the mitochondria and the granular endoplasmic reticulum were observed after incubation in TCBZ.SO alone, together with reduced secretory activity and evidence of autophagy. However, these changes were far more pronounced in combination-treated flukes. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

Effect of the P-glycoprotein inhibitor, R(+)-verapamil on the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by the inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R-VPL]. In the first experiment, flukes were initially incubated for 2 h in R-VPL (100 μM), then incubated for a further 22 h in R-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg/ml, or 0.1327 μM). For controls, flukes were incubated for 24 h in R-VPL and TCBZ.SO on their own. In a second experiment, flukes were removed from the incubation media following cessation of movement. In the third experiment, Sligo flukes were incubated in lower concentrations of R-VPL (10 μM) and TCBZ.SO (15 μg/ml, or 0.0398 μM). Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R-VPL alone had minimal effect on either isolate. After treatment with TCBZ.SO alone, there was greater surface disruption to the Cullompton than Sligo isolate. However, combined treatment of R-VPL+TCBZ.SO led to more severe surface changes to the Sligo isolate than with TCBZ.SO on its own; this potentiation of drug activity was not seen with the Cullompton isolate. The phenomenon was evident at both concentrations of TCBZ.SO. Inclusion of R-VPL in the incubation medium also reduced the time taken for the flukes to become inactive; again, this effect was more distinct with the Sligo isolate. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

Increased susceptibility of a triclabendazole (TCBZ)-resistant isolate of Fasciola hepatica to TCBZ following co-incubation in vitro with the P-glycoprotein inhibitor, R(+)-verapamil

A study was carried out to investigate whether the action of triclabendazole sulphoxide (TCBZ.SO) against the liver fluke, Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for this in vitro study and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. For experiments with the Oberon isolate, flukes were incubated for 24 h with either R(+)-VPL (1×10-4 m) on its own, TCBZ.SO (15 μg mL-1) alone, a combination of R(+)-VPL (1×10-4 m) plus TCBZ.SO (15 μg mL-1), TCBZ.SO (50 μg mL-1) on its own, or a combination of TCBZ.SO (50 μg mL-1) plus R(+)-VPL (1×10-4 m). They were also incubated in TCBZ.SO (50 μg mL-1) alone or in combination with R(+)-VPL (1×10-4 m) until they became inactive; and in TCBZ.SO (50 μg mL-1) alone for a time to match that of the combination inactivity time. Flukes from the Cullompton isolate were treated with either TCBZ.SO (50 μg mL-1) alone or in combination with R(+)-VPL (1×10-4 m) until they became inactive, or with TCBZ.SO (50 μg mL-1) alone time-matched to the combination inactivity time. Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R(+)-VPL alone had a minimal effect on either isolate. TCBZ.SO treatment had a relatively greater impact on the TCBZ-susceptible Cullompton isolate. When R(+)-VPL was combined with TCBZ.SO in the incubation medium, however, the surface disruption to both isolates was more severe than that seen after TCBZ.SO treatment alone; also, the time taken to reach inactivity was shorter. More significantly, though, the potentiation of drug activity was greater in the Oberon isolate; also, it was more distinct at the higher concentration of TCBZ.SO. So, the Oberon isolate appears to be particularly sensitive to efflux pump inhibition. The results of this study suggest that enhanced drug efflux in the Oberon isolate may be involved in the mechanism of resistance to TCBZ.

Disruption of vitellogenesis and spermatogenesis by triclabendazole (TCBZ) in a TCBZ-resistant isolate of Fasciola hepatica following incubation in vitro with a P-glycoprotein inhibitor

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. In the first experiment, flukes were initially incubated for 2 h in R(+)-VPL (100 μ m), then incubated in R(+)-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg mL-1, or 133·1 μ m) until flukes ceased movement (at 9 h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15 h). In the third experiment, flukes were incubated for 24 h in R(+)-VPL on its own. Changes to the testis tubules and vitelline follicles following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the morphology of the two tissues. Greater disruption was observed when the drugs were combined, in terms of the block in development of the spermatogenic and vitelline cells and the apoptotic breakdown of the remaining cells. Sperm formation was severely affected and abnormal. Large spaces appeared in the vitelline follicles and synthesis of shell protein was disrupted. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.