Purification of Adult Hymenolepis diminuta (Cestoda) Mitochondrial NADPH→NAD+ Transhydrogenase.

The mitochondrial, inner-membrane-associated, reversible NADPH→NAD+ transhydrogenase of the energetically anaerobic adult cestode Hymenolepis diminuta connects NADPH generation, via a mitochondrial NADP+-specific "malic" enzyme, with NADH formation needed for electron transport. In reducing the pyridine nucleotide, the enzyme concomitantly catalyzes transmembrane proton translocation, thereby coupling NADH formation to ATP generation or NADPH formation to ATP hydrolysis. Detergent-solubilized transhydrogenase, from isolated mitochondrial membranes, was purified to apparent homogeneity using ion exchange and hydroxylapatite chromatographies. The enzyme displayed a monomeric Mr of ∼110 kDa and required phospholipid, without which activity was rapidly lost. Of the phospholipids examined, phosphatidylcholine was the most effective. Transhydrogenase-catalyzed NADH formation was inhibited by NAD(P)+ and adenylates, suggesting regulatory effects of the pyridine nucleotides and effects of pyridine nucleotide-simulating molecules. In keeping with its proton-translocating function, the enzyme was inhibited by dicyclohexylcarbodiimide. The isolated enzyme catalyzed neither NADH→NADP+ nor NADH→NAD+ transhydrogenations, thereby suggesting a need for a minimal coupling to electron transport for the NADH→NADP+ reaction as well as enzyme specificity. Anti-transhydrogenase monospecific antibodies proved inhibitory to NADPH→NAD+ transhydrogenation catalyzed by both isolated and membrane-associated enzymes. This purification study apparently represents a first for parasitic helminths or multicellular invertebrates generally and establishes a framework for evaluating the transhydrogenase as a potential site for specific chemotherapeutic attack.

The activity of drug-metabolizing enzymes and the biotransformation of selected anthelmintics in the model tapeworm Hymenolepis diminuta.

The drug-metabolizing enzymes of some helminths can deactivate anthelmintics and therefore partially protect helminths against these drugs' toxic effect. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (albendazole, flubendazole, mebendazole) in the rat tapeworm Hymenolepis diminuta, a species often used as a model tapeworm. In vitro and ex vivo experiments were performed. Metabolites of the anthelmintics were detected and identified by HPLC with spectrofluorometric or mass-spectrometric detection. The enzymes of H. diminuta are able to reduce the carbonyl group of flubendazole, mebendazole and several other xenobiotics. Although the activity of a number of oxidation enzymes was determined, no oxidative metabolites of albendazole were detected. Regarding conjugation enzymes, a high activity of glutathione S-transferase was observed. A methyl derivative of reduced flubendazole was the only conjugation metabolite identified in ex vivo incubations of H. diminuta with anthelmintics. The results revealed that H. diminuta metabolized flubendazole and mebendazole, but not albendazole. The biotransformation pathways found in H. diminuta differ from those described in Moniezia expanza and suggest the interspecies differences in drug metabolism not only among classes of helminths, but even among tapeworms.

Hymenolepis diminuta: experimental studies on the antioxidant system with short and long term infection periods in the rats.

Many helminths cause long-lasting infections, living for several years in mammalian hosts reflecting a well balanced coexistence between host and parasite. There are many possible explanations as to how they can survive for lengthy periods. One possibility is their antioxidant systems, which can serve as defence mechanisms against host-generated oxygen radicals. Therefore, the aim of this experimental study was to examine the antioxidant system in Hymenolepisdiminuta during short (1.5 months young tapeworms) and long (1.5 years old tapeworms) term infection in the rat small intestine. The strobilae of H. diminuta tapeworms (14 young and three old) were divided into three pieces: the anterior part, containing the genital primordiae in the immature segments; the medial part, containing the early uterus in the mature, hermaphroditic proglottids and the terminal part with the mature gravid uterus in the gravid segments. Supernatants of these fragments were used for determination of markers of oxidative stress: concentration of thiobarbiturate reactive substances (TBARS) and of reduced glutathione (GSH), and the activity of antioxidant enzymes: superoxide dismutase (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (GSHPxs), glutathione transferase (GST) and glutathione reductase (GSHR). The results indicated changes in levels of oxidative stress markers and antioxidant enzyme activity in both the young and old forms of H. diminuta. Relatively high activity of SOD (particularly in the anterior part of young tapeworms) was observed, as was increased activity of total GSHPx and a relatively high concentration of GSH in all parts of the tapeworms. These are caused by exposure to increased amount of ROS, which are produced during the inflammatory state. Due to the high activity of antioxidant enzymes, the anterior section of young and old tapeworms is equipped with a very effective antioxidant system. Old organisms also effectively resist oxidative stress due to reduced levels of lipid peroxidation and the high activity of GST, all of which suggest good adaptation to the hostile environment in the host's intestine.

Transhydrogenase and the anaerobic mitochondrial metabolism of adult Hymenolepis diminuta.

The adult cestode, Hymenolepis diminuta, is essentially anaerobic energetically. Carbohydrate dissimilation results in acetate, lactate and succinate accumulation with succinate being the major end product. Succinate accumulation results from the anaerobic, mitochondrial, 'malic' enzyme-dependent utilization of malate coupled to ATP generation via the electron transport-linked fumarate reductase. A lesser peroxide-forming oxidase is apparent, however, fumarate reduction to succinate predominates even in air. The H. diminuta matrix-localized 'malic' enzyme is NADP-specific whereas the inner membrane (IM)-associated electron transport system prefers NADH. This dilemma is circumvented by the mitochondrial, IM-associated NADPH-->NAD+ transhydrogenase in catalyzing hydride ion transfer from NADPH to NAD+ on the IM matrix surface. Hydride transfer is reversible and phospholipid-dependent. NADP+ reduction occurs as a non energy-linked and energy-linked reaction with the latter requiring electron transport NADH utilization or ATP hydrolysis. With NAD+ reduction, the cestode transhydrogenase also engages in concomitant proton translocation from the mitochondrial matrix to the intermembrane space and supports net ATP generation. Thus, the cestode NADPH-->NAD+ system can serve not only as a metabolic connector, but an additional anaerobic phosphorylation site. Although its function(s) is unknown, a separate IM-associated NADH--> NAD+ transhydrogenation, catalyzed by the lipoamide and NADH dehydrogenases, is noted.

Hymenolepis diminuta: mitochondrial transhydrogenase as an additional site for anaerobic phosphorylation.

Employing adult Hymenolepis diminuta SMP and exogenous pyridine nucleotide-generating systems, reduced pyridine nucleotide-dependent net 32P incorporation into ATP was examined. NADH supported rotenone-sensitive 32P incorporation and this rate increased markedly with fumarate addition, in keeping with an active fumarate reductase. Interestingly, corresponding evaluations with NADPH did not result in detectable phosphorylation in the absence or presence of fumarate. However, with NAD addition, but without NAD generation, active NADPH-dependent phosphorylation occurred, thereby demonstrating mitochondrial transhydrogenase involvement, and 32P incorporation increased significantly with fumarate addition. More importantly, in the presence of rotenone and both NADPH and NAD generation, significant net 32P incorporation was noted, but was undetectable in the presence of DCCD or protonophores (e.g., niclosamide). Without NAD generation, minimal phosphorylation occurred. These data demonstrate that with ongoing NADPH and NAD generation, the H. diminuta, proton-translocating, mitochondrial transhydrogenase can serve as an additional anaerobic phosphorylation site. A model is presented.