Fasciola hepatica: ultrastructural changes to the tegument of juvenile flukes following incubation in vitro with the deacetylated (amine) metabolite of diamphenethide.

Ultrastructural changes to the tegument of 5-week-old, 3-week-old and freshly-excysted Fasciola hepatica following in vitro incubation with the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 microgramsml-1) were examined by transmission electron microscopy. A similar sequence of tegumental changes occurred in all three age groups of fluke, although, with increasing fluke age, the time before onset increased and the damage became more extensive. The 5-week-old flukes showed an initial stress response after 3 h, typified by blebbing of the apical plasma membrane, formation of microvilli and an accumulation and accelerated release of secretory bodies at the tegumental apex, as well as swelling of the basal infolds. The swelling increased in extent with progressively longer periods of incubation in DAMD, leading to extreme edema and sloughing of the tegument after 9 h. The 3-week-old flukes showed a stress response and swelling of the basal infolds after only 1.5 h, although sloughing of the tegument did not occur until after 9 h. In the freshly-excysted metacercaria, a stress response and some sloughing of the tegument were evident after only 0.5 h. At all stages of development, the ventral tegument was more severely affected than the dorsal. Changes also occurred to the tegumental cells which were indicative of a disruption in the synthesis and release of tegumental secretory bodies: the amount of GER became reduced, the cisternae became swollen and their ribosomal covering decreased, the Golgi complexes disappeared from the cells and the numbers of secretory bodies in the cells also decreased. The heterochromatin content of the nuclei increased and eventually the tegumental cells began to break down. Again, the changes became apparent more rapidly at the earlier stages of development. The ultrastructural changes to the tegument are linked to a possible mode of action for diamphenethide as an inhibitor of protein synthesis. In turn, the results may help to explain the drug's high efficacy against juvenile stages of F. hepatica.

[Manifestation of the mutagenic effect of acemidophen depending on its metabolism in various mammalian species (acemidophen--a promutagen)]. / Proiavlenie mutagennogo éffekta atsemidofena v zavisimosti ot ego metabolizma u raznykh vidov mlekopitaiushchikh (atsemidofen--promutagen).

While studying the specific variability of metabolism of the fasciolacide anthelmintic acemidophen, it is found that it possesses no mutagenic activity. This activity is peculiar to its metabolite-free amine L2, formed in mice organism by deacetylation. Thus, acemidophen is shown to be promutagen.

Fasciola hepatica: tegumental surface alterations following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide.

The effect of the deacetylated (amine) metabolite of diamphenethide (10 micrograms/ml) on the tegumental surface of Fasciola hepatica over a 24 h period in vitro has been determined by scanning electron microscopy. Blebbing begins around the oral sucker after 3 h and then passes backwards along the body, reaching the ventral sucker and midbody by 6 h, and finally the posterior end of the body (by 12 h). Initially, the blebs are small, the tegument surrounding the spines is swollen and the tegument generally has a smooth, swollen appearance. This submerges the spines below the body surface. At higher magnification the surface is seen to bear microvillous-like projections in addition to the blebs and surface pitting is deeper than normal. Later on, the blebs increase in size and burst, causing lesions and loss of spines. Lesions begin to appear on the oral cone and ventral sucker after 6 h, in the midbody by 12 h and on the dorsal surface of the posterior region after 24 h. By this time the damage is extensive: around the oral and ventral suckers, and over large areas of the oral cone and midbody region the tegument has been stripped off to expose the basal lamina beneath. The dorsal surface of the fluke is consistently more severely affected than the ventral surface.

Fasciola hepatica: tegumental changes induced in vitro by the deacetylated (amine) metabolite of diamphenethide.

The effect of the deacetylated (amine) metabolite of diamphenethide (10 mugm/ml) on the tegument of Fasciola hepatica over a 24 hr period in vitro has been determined by means of transmission electron microscopy. In the tegumental syncytium, there is an initial accumulation of T2 secretory bodies at the apical surface (after 6 hr), together with increased exocytosis of secretory bodies and blebbing of the surface membrane. After 9 hr, the two surfaces of the fluke show different tegumental responses to drug treatment with a marked swelling of the basal infolds in the dorsal tegument, while the ventral tegument remains normal. By 18 hr, the swelling in the dorsal tegument is very severe, the entire basal region becoming edematous. In some areas, the tegument becomes detached to expose the basal lamina. The ventral tegument retains a fairly normal morphology, although there is a slight swelling of the basal infolds. The edema spreads internally to the cell bodies, beginning after 9 hr on the dorsal side of the fluke and 18 hr on the ventral side. By 18 hr, the flooding on the dorsal side is very severe and the cells attenuated, retaining few contacts with the surrounding parenchyma. From 9 hr onwards, there are progressive changes in cell structure, including a decrease in amount of granular endoplasmic reticulum and extent of its ribosomal covering, a decrease in numbers of secretory bodies, a swelling of the trans-most Golgi cisternae and disruption of the release of secretory bodies, and a swelling and disorganization of the mitochondria. The results are discussed in relation to the postulated activity of the deacetylated (amine) metabolite of diamphenethide as a Na+ ionophore.

Studies of the effect of diamphenethide and oxyclozanide on the metabolism of Fasciola hepatica.

Studies were made of the effects of diamphenethide-amine on glucose transport, glycogen breakdown, adenine nucleotides, metabolites and excretory products in both parenchymal and bile duct flukes in vitro and in bile duct flukes in vivo. The most consistent and pronounced effect observed was an elevation of malate levels. There appeared to be no differences between responses of parenchymal and of bile duct flukes to diamphenethide-amine. For comparative purposes the effect of oxydozanide was assessed on most of these parameters in bile duct flukes in vitro. Not all the changes caused by oxyclozanide were characteristic of an uncoupler; however, the pattern of changes in the metabolites was markedly different from that caused by diamphenethide-amine.

Protection of Fasciola hepatica against flukicidal action of the amine of diamphenethide in vitro.

A variety of compounds structurally related to serotonin and dopamine were tested for their ability to protect adult liver flukes against the flukicidal effect in vitro of an active metabolite of diamphenethide (diamphenethide-amine). The most effective was dopamine. The protective effect of dopamine was also assessed by analysis of the metabolite and adenine nucleotide levels and excretory products of flukes exposed to diamphenethide-amine in vitro. Most notably, dopamine appeared to depress considerably the large elevation of internal malate which is a characteristic effect of diamphenethide-amine on liver flukes.